Expression of the Pycnoporus cinnabarinus laccase geneof the recombinant enzyme Eric Record1, Peter J.. The identity of the recombinant protein was further confirmed by immunodetection u
Trang 1Expression of the Pycnoporus cinnabarinus laccase gene
of the recombinant enzyme
Eric Record1, Peter J Punt2, Mohamed Chamkha3, Marc Labat3, Cees A M J J van den Hondel2
and Marcel Asther1
1
Unite´ INRA de Biotechnologie des Champignons Filamenteux, IFR-IBAIM, Universite´s de Provence et de la Me´diterrane´e, ESIL, Marseille, France;2Department 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 cinnabarinuslaccase lac1 gene was overexpressed
in Aspergillus niger, a well-known fungal host producing a
large amount of homologous or heterologous enzymes for
industrial applications The corresponding cDNA was
placed under the control of the glyceraldehyde-3-phosphate
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 secretion of laccase into the
culture medium as an active protein, but the A niger
pre-prosequence allowed an 80-fold increase in laccase
produc-tion The identity of the recombinant protein was further
confirmed by immunodetection using Western blot analysis
and N-terminal sequencing The molecular 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 purified in a three-step procedure including a fractionated precipitation using ammonium sulfate, and a concentration by 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 cinnabarinus; heterologous expression; Aspergillus niger; fungal
Laccases (p-diphenol:O2 oxidoreductase; EC 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 demonstrated in fungi, plants and more recently 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 heterogeneous 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 the protein molecular mass and laccases exhibit
1–4 isozymes [7] The optimum pH varies from 3 to 6
depending on the substrate [8] They are 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 mammalian plasma protein ceruloplasmin 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 domain (type-2 and type-3 coppers) is responsible of the adsorption of light at 330 nm The T1 copper domain is 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, the trinuclear center, which is the dioxygen-binding site, accepts these electrons with the concomitant reduction of the molecular oxygen This three-step process allows the oxidation of phenolic com-pounds, including polyphenols, methoxy-substituted mon-ophenols, aminophenols and a considerable range of other compounds [7] Metal ions, such as Fe2+, and many nonphenolic compounds, such as ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate]) are oxidized by laccases [12] The biological function of most laccases is yet unclear They have been indicated to be involved in pigment formation, lignin degradation and detoxification [7] Never-theless, laccases are very interesting tools for industrial applications, i.e for bleaching in pulp and paper indus-tries, for detoxification of recalcitrant biochemicals, for
Correspondence to E Record, Unite´ INRA de Biotechnologie des
Champignons Filamenteux, IFR-IBAIM, Universite´s de Provence et
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:
record@esil.univ-mrs.fr?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 November 2001, accepted 20
November 2001)
Trang 2bioconversion of chemicals or treatment of beverages in
agrochemical industry [3]
In our laboratory, we demonstrated, the presence of two
isozymes, LacI and LacII, in the white-rot fungus
Pycno-porus cinnabarinusstrain ss3, which is the monokaryotic
strain derived from the dikaryotic parental strain I-937 [13]
The gene encoding the laccase LacI was isolated and its
expression characterized (GenBank accession number
AF170093) The laccase 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 recombinant protein in Pichia
was high enough to allow the first structure function studies,
but too low to consider industrial approaches In order 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
E X P E R I M E N T A L P R O C E D U R E S
Strains, culture media
Escherichia coliJM109 (Promega, Charbonnieres, France)
was used for construction and propagation of vectors
A nigerstrain D15#26 (pyrg–) [15] was used for
hetero-logous expression After cotransformation with vectors
containing, respectively, the pyrG gene and the laccase
cDNA, A niger was grown on selective solid minimum
medium (without uridine) containing 70 mMNaNO3, 7 mM
KCl, 11 mMKH2HPO4, 2 mMMgSO4, glucose 1% (w/v),
and trace elements (1000· stock solution consists of: 76 mM
ZnSO4, 178 mM H3BO3, 25 mM MnCl2, 18 mM FeSO4,
7.1 mMCoCl2, 6.4 mMCuSO4, 6.2 mMNa2MoO4, 174 mM
EDTA)
Chemicals Restriction enzymes and Pfu DNA polymerase were, respectively, purchased from Life Technologies (Cergy Pontoise, France) and Promega [a-32P]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 primer sequences Constructs A and
pLac1-B contained the laccase cDNA corresponding to the laccase gene, lac1 from P cinnabarinus (GenBank accession no AF 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 gene (gpdA) promoter, the 5¢ untrans-lated region of the gpdA mRNA, and the A nidulans trpC terminator were used to drive the expression of the laccase encoding sequence
Aspergillus transformation and laccase production Fungal cotransformation was basically carried out as described by Punt & van den Hondel [16] using each of the laccase expression vectors and pAB4-1 [17] containing the pyrG selection marker, in a 10 : 1 ratio Transformants were selected for uridine prototrophy Cotransformants containing expression vectors were selected as described in the following section
In order to screen the laccase production in liquid medium, 50 mL of culture medium containing 70 mM NaNO3, 7 mM KCl, 200 mM Na2HPO4, 2 mM MgSO4,
Table 1 Cloning strategy For each expression vector are indicated the name of the primers used for amplification of the laccase cDNA and addition of cloning sites, recipient Aspergillus expression vector and restriction sites used in the final cloning procedure.
Expression
vectors
Primers
Cloning vectors
Cloning site restriction fragments
Cloning site vectors Forward Reverse
a EMBL accession number Z32701; b EMBL accession number Z32750.
Table 2 Oligonucleotides used for cDNA amplification and cloning St, stop codon Restriction sites are underlined.
Trang 3glucose 10% (w/v), trace elements and adjusted to pH 5
with a 1-M citric acid solution were inoculated by
1· 106sporesÆmL)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-Mcitric
acid For protein purification, 850-mL cultures were
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 lM 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 lMABTS at
420 nm to the respective radical (e420 ¼ 36 mM )1Æcm)1)
[18], in the presence of 50 mM sodium tartrate pH 4.0 at
30°C (standard conditions) For the stability to the pH or
the optimal pH determination, syringaldazine (17 lM) was
also used as the substrate by monitoring the production of
colored quinone at 530 nm (e530 ¼ 65 mM )1Æcm)1) [6]
Activity is indicated in international units (IU) which are the
amount of laccase that oxidizes 1 lmol of substrate 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 mAÆcm)2at 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 assay [20]
Northern blot analysis
Total RNA was isolated at various time from biomass
aliquots of A niger as indicated by Wessels et al [21] An
aliquot of 15 lg of total RNA was denatured at 65°C in a
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 min (0.6 JÆcm)1Æmin)1) The blots were probed with a32P-labelled probe consisting of the laccase cDNA and for loading control a 18S PCR amplified DNA was used as a probe Blotted membranes were hybridized overnight at 65°C in a buffer containing 0.5M sodium phosphate buffer pH 7.2 with 0.01M EDTA, 7% (w/v) SDS, and 2% (w/v) blocking reagent (Roche Molecular Biochemicals, Meylan, France) The most stringent posthy-bridization wash consisted of a 2· 15 min in 0.2 · NaCl/ Cit (NaCl/Cit 20·: 0.3M sodium citrate buffer pH 7.0, with 3M 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 mL 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 10 kDa) (Millipore) The medium was further concentrated by a two-step ammonium 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 with ammonium sulfate and stirred for 2 h at 4°C The precipitate was collected by centrifugation at 13 000 g for 30 min and dissolved in 4 mL of buffer A (25 mM 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 and 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 gradient (0–500 mMin buffer A) at a flow rate
of 1 mLÆmin)1 and collected with fractions of 1 mL Laccase activity was eluted (3 mL) with fractions corre-sponding to 350 mMNaCl and dialyzed against buffer A Characterization of the recombinant laccase
Protein analysis Protein concentration was determined according to Lowry et al [23] with bovine serum albumin as standard Protein purification was followed by SDS/PAGE
on 10% polyacrylamide slab gels [19] Proteins were stained with Coomassie blue Analytical isoelectric focusing was performed with 2.5–5.0 gradient gels using a Pharmacia LKB Phastsystem (Amersham Pharmacia 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 For an explanation, see
Experimental procedures and Table 1.
Trang 4Temperature 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 various 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 purified
laccase in 50 mMcitrate/100 mMphosphate buffer (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 UÆmL)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 mM citrate/
100 mM phosphate buffer (pH 2.5–7.0) and in 50 mM
phosphate buffer (pH 6–8) at 30°C ABTS was used as
the substrate in both experiments and syringaldazine for
optimal pH determination
R E S U L T S
Transformation and screening
In a cotransformation experiment, A niger D15#26 was
transformed with a mixture of plasmid pAB4-1 and each of
the two expression vectors containing the laccase cDNA
from P cinnabarinus Transformants 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 with ABTS Recombinants
expressing laccase were identified by the appearance of a
green zone around the colonies after 7–10 days at 30°C
Colored zones on plates were not observed in the case of
control transformants lacking the laccase cDNA Thirty
positive clones were cultured in liquid for each construction
and then assayed at optimal day of production Results for
laccase activity were ranging from 30–90 IUÆL)1(day 7) and
from 1800–7000 IUÆL)1(day 10), respectively, for A niger
transformed by pLac1-A and pLac1-B The best clone was
selected for each construction in order to study the time
course of the laccase activity
Study of the recombinant laccase production
inA niger
For both expression vectors, the laccase activity was found
in the culture medium, indicating that laccase was secreted
from A niger Activity was not found in the control culture
(transformation with pAB4-1, without pLac1) In both
cultures, mycelial dry weight increased until day 5, and
reached a maximum of 17–18 gÆL)1until 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)1and was
more or less stable until day 12 Using the GLA signal 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 inA niger Production of the recombinant laccase for the 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 cinnabarinuslaccase The Western blot analysis showed
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 12 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 of A niger by three successive steps (Table 3) Eight hundred and fifty millilitres of medium
0 5 10 15 20
0 5 10 15 20
0 50 100 150
0 5000 10000 A
B
1 )
1 )
1 )
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 plotted as a function of time for pLac1-A (A) and pLac 1-B (B).
Trang 5were concentrated 6.3-fold by ultrafiltration with a recovery
of 94%, then further concentrated by a two-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 of 16%,
yielding 6.3 mg of laccase
Molecular mass and isoelectric point The homogeneity of
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 the 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 was
100 min, whereas at 75 °C, the laccase was completely inactivated in less than 15 min pH stability was studied between pH 2.5 and 5.0 and results showed that the recombinant laccase was stable at pH 5.0 for at least
120 min Below pH 5.0, the laccase activity decreased 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°C and 70 °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 mMand was estimated to be 55 lM
D I S C U S S I O N
White-rot fungi that degrade lignin and 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 the end (exo-glucanases, cellobiohydrolases) or in the middle (endo-glucanases) of a chain and then
b-glyco-Sd 1 b-glyco-Sd 2
94 kDa
67 kDa
43 kDa
30 kDa
20 kDa
Fig 3 SDS/PAGE gel and Western blot analysis of the laccase
pro-duction in the P cinnabarinnus culture medium Sd, molecular mass
standards; SDS/PAGE stained with Coomassie blue (lane 1) and
Western blot (lane 2) analysis of the culture medium For
immuno-detection, antibodies raised 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)
Fig 4 Northern 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 the probe The 18S PCR amplified DNA was used as the loading control.
Trang 6sidases 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 perper-oxidase were detected in lignin
degradation conditions [26] For these reasons, we studied
P cinnabarinusas a model to 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 the
Deuteromycete A niger The recombinant laccase was also
purified to homogeneity and physico-chemically
character-ized in order to compare it’s properties to those of the
wild-type protein
Two expression vectors were constructed containing the cDNA encoding the P cinnabarinus laccase either with its own signal peptide or fused with the GLA preprosequence 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, we have cloned and expressed
P cinnabarinuslaccase 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 8 mgÆL)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. 5 mgÆL)1, though filamentous fungal hosts allowed a production of
0
20
40
60
80
100
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) and 75 °C (+) Five hundred l M ABTS was used
as the substrate for enzyme assay.
0 20 40 60 80 100
0 10 20 30 40 50 60 70 80 90
Temperature (°C) Fig 7 Effect of the temperature on the activity of the purified laccase Various temperatures in the range of 25 °C to 85 °C were tested with
500 l M ABTS as the substrate.
0 20 40 60 80 100
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) and 17 l M of syringaldazine (j) as the substrate.
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 and lane 1, pure recombinant laccase stained with
Coomassie blue.
Trang 710–20 mgÆL)1 The best production of recombinant laccase
was recently obtained with the Coprinus cinereus laccase
gene expressed in A oryzae where results reached from 8 to
135 mgÆL)1 [31] In conclusion, P cinnabarinus laccase
production in A niger was quite satisfactory 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 All the main characteristics of the
recom-binant enzymes, i.e molecular mass, pI, optimal
temper-ature and pH, stability to the tempertemper-ature, N-terminal
sequence and the Michaelis constant, were compared to
those of the P cinnabarinus laccase (data not shown)
N-Terminal sequence, molecular mass, and pI, are
iden-tical for both proteins, i.e 70 kDa; pI around 3.7 The Km
for ABTS was estimated to be 55 lM for the native and
the recombinant protein The optimal temperature varies in
the range of 65–70°C, and optimal pH is 4 for both
proteins In addition, the 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 molecular 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 also described for the Trametes villosa laccase
produced in A oryzae [6] Glycosylation was 0.5% of
the molecular mass of the native laccase and and 10% for
the recombinant laccase In the heterologous production
of the P cinnabarinus laccase in P pastoris [14] or the
T villosalaccase 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, the 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 to
improve the productivity and subsequently obtain large
protein amounts for industrial applications
A C K N O W L E D G E M E N T S
This research was supported by the European program, Quality of Life
and Management of Living Resources (PELAS: (Peroxidases and
Laccases) Fungal metalloenzymes oxidizing aromatic compound of
industrial interest) as well as GIS-EBL (Conseil Re´gional
Provence-Alpes-Coˆte d’Azur and Conseil Ge´ne´ral 13, France) We thank
Jean-Luc Robert for technical assistance in enzymatic assays.
R E F E R E N C E S
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