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: 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 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. 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Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme Eric Record 1 ,. 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