Protein Expression and PuriWcation 37 (2004) 126–133

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Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme

Anthony Levasseur,¤ Isabelle Benoit, Michèle Asther, Marcel Asther, and Eric Record

UMR 1163 INRA de Biotechnologie des Champignons Filamenteux, IFR-BAIM, Universités de Provence et de la Méditerranée, ESIL,163 avenue de Luminy, Case Postale 925, 13288 Marseille cedex 09, France

Received 2 March 2004, and in revised form 28 May 2004Available online 8 July 2004

Abstract

The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeBgene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of thegpd promoter and trpC terminator and expressed in a protease deWcient A. niger strain. Homologous overproduction allows to reachan esterase activity of 18 nkat mL¡1 against MCA as substrate. The improvement factor was 16-fold higher as compared to the pro-duction level obtained with non-transformed A. niger strain induced by sugar beet pulp. The corresponding secretion yield was esti-mated to be around 100 mg L¡1. Recombinant FAEB was puriWed 14.6-fold to homogeneity from an 8-day-old culture by a singleaYnity chromatographic step with a recovery of 64%. SDS–PAGE revealed a single band with a molecular mass of 75 kDa, whileunder non-denatured conditions, native enzyme has a molecular mass of around 150 kDa conWrming that the recombinant FAEB isa homodimer. The recombinant and native FAEB have the same characteristics concerning temperature and pH optima, i.e., 50 °Cand 6, respectively. In addition, the recombinant FAEB was determined to be quite stable up to 50 °C for 120 min. Kinetic constantsfor MCA, MpCA, and chlorogenic acid (5-O-caVeoyl quinic acid) were as follows: Km: 0.13, 0.029, and 0.16 mM and Vmax: 1101,527.6, and 28.3 nkat mg¡1, respectively. This is the Wrst report on the homologous overproduction of feruloyl esterase B in A. niger. 2004 Elsevier Inc. All rights reserved.

Plant cell wall is composed of polysaccharides andlignin embedded in a complex and organized structure.This physical biobarrier constitutes an important protec-tion against microbial invasion. This structure isstrengthened by a close interaction between cellulosemicroWbrills and hemicellulose or pectin polysaccha-rides. Additional cross-linkages such as diferulic acidbridges between hemicellulose adjacent chains [1] orbetween lignin and hemicellulose [2] increase the com-plexity of the cell wall structure and contribute toimprove its resistance to hydrolysis. Ferulic acid andother aromatic compounds are present as terminal sidegroups in xylan and pectin but with diVerent polysaccha-ride sites of attachment. In xylan, ferulic acid is attachedto O5 of terminal arabinose residue [3], whereas in the

¤ Corresponding author. Fax: +33-4-91-82-86-01.E-mail address: anthony.levasseur@esil.univ-mrs.fr (A. Levasseur).

1046-5928/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.pep.2004.05.019

pectic hairy regions, this aromatic compound is linked toO2 of arabinose residues or O6 of galactose residues [4].

Plant saprophytic micro-organisms produce a pleth-ora of enzymes to degrade plant cell wall and use the cellwall components as nutrients. Some micro-organismshave evolved enzymes such as esterases to release aro-matic acids and to allow a facilitated accessibility ofmain-chain degrading enzymes to the polysaccharidebackbone (for a review, see [5,6]). In Aspergillus niger,two diVerent feruloyl esterases were puriWed from theliquid culture when the strains were grown on oat speltsxylan or sugar beet pulp [7,8]. Their corresponding genes(faeA and faeB) were cloned and their regulation wasstudied using inducers such as a range of aromatic com-pounds, polysaccharides or diVerent carbon sources [9–12]. Expression proWles of both genes indicated distinctactivating mechanisms because diVerent aromatic com-pounds were able to induce preferentially a particulargene. However, the two esterase genes were commonly

A. Levasseur et al. / Protein Expression and PuriWcation 37 (2004) 126–133 127

controlled by the carbon catabolite repression mediatedby the DNA-binding repressor CreA when easily metab-olizable carbon sources are present [10,11,13].

Potential applications of feruloyl esterases are multi-ple. Indeed, ferulic acid, found in agricultural by-prod-ucts, is an attractive industrial compound by virtue of itsantioxidant, antimicrobial, and photoprotectant proper-ties [14] or its potential biotransformation to vanillin asa food Xavour precursor [15]. Synergistic actions havebeen already observed between feruloyl esterase A(FAEA) from A. niger and endoxylanases that improvethe ferulic acid release and could be useful to producesuYcient amount of these cinnamic acids for applica-tions [16–18]. In addition, the FAEB is more active forferulic acid release towards sugar beet pectin thanFAEA, whereas the opposite was observed with wheatarabinoxylan [11]. Depending on the agricultural by-product to treat, a particular feruloyl esterase fromA. niger must be used for an eYcient ferulic acid release.

Recently, in the pulp and paper sector, FAEA wastested for the Wrst time in wheat straw pulp bleachingprocedure and a sequential treatment with xylanaseand laccase yielded a very eYcient deligniWcationclose to 75% [19]. This biotechnological process shouldavoid to employ currently used chemical treatments thatlead to high level of polluting agents such as chlorolig-nin. Production yields of these feruloyl esterases fromwild-type strain are generally weak and remain the usualbottleneck for an industrial application. Moreover, cin-namic acids, known to be good inducers of feruloylesterase genes, are expensive compounds and their addi-tion in the culture medium is not economically viable. Asa general rule, the valorization of polluting by-productfrom agricultural wastes or the pulp bleaching applica-tions need to obtain fast and high quantities of enzyme.

In this paper, we describe the cloning and the homol-ogous overexpression of the second major feruloyl ester-ase faeB gene from A. niger. The physico-chemicalproperties of the recombinant protein were compared tothose of the native enzyme. This overproduction systemis a prerequisite to produce large-scale FAEB for struc-ture–function studies and for biotechnological applica-tions in the food industry or in the pulp and papersector.

Methods

Strains, culture media

Escherichia coli JM109 (Promega, Charbonnières,France) was used for construction and propagation ofvectors and Aspergillus niger strain D15#26 (pyrg¡) [20]for homologous expression of the feruloyl esterase Bgene (faeB) [11]. After cotransformation with vectorscontaining, respectively, the pyrG gene and the feruloyl

esterase gene, A. niger was grown on selective solid mini-mal medium (without uridine) containing 70 mM NaNO3,7 mM KCl, 11 mM KH2HPO4, 2 mM MgSO4, glucose 1%(w/v), and trace elements [1000£ stock: 76 mM ZnSO4,25 mM MnCl2, 18 mM FeSO4, 7.1 mM CoCl2, 6.4 mMCuSO4, 6.2 mM Na2MoO4, and 174 mM ethylenediamine-tetraacetic acid (EDTA)]. A. niger BRFM1311 (Banque deRessources Fongiques de Marseille) was used for genomicDNA extraction and the resulting DNA was used astemplate for PCR ampliWcation strategy.

Chemicals

Restriction enzymes and Pfu DNA polymerase were,respectively, purchased from Invitrogen (Cergy Pon-toise, France) and Promega. DNA sequencing was per-formed by Genome Express (Grenoble, France). CaVeicacid, p-coumaric acid were obtained from Apin Chemi-cals (Oxon, UK). 5-O-CaVeoyl quinic acid was fromSigma (St. Quentin Fallavier, France).

Expression vectors

Expression vector was constructed using a PCR clon-ing approach, and the cloned PCR products werechecked by sequencing. Genomic DNA was used as tem-plate for PCR ampliWcation with two speciWc primersdesigned from the available faeB sequence (GenBankAJ309807), i.e., an upstream primer 5�-acg tca tga aagtag caa gtc tcc-3� and a downstream primer 5�-tac aag ctttta gtg gtg gtg gtg gtg gtg tac gca agt cca gga gtt agg-3�containing “BspHI” and “HindIII” restriction sites,respectively. The PCR product was then cloned in theexpression vector pAN52.3 (EMBL Accession No.Z32689), to obtain the faeB expression vector. In thisvector, the Aspergillus nidulans glyceraldehyde-3-phos-phate dehydrogenase gene (gpdA) promoter, the 5�untranslated region of the gpdA mRNA, and theA. nidulans trpC terminator were used to drive theexpression of the FAEB encoding sequence.

Aspergillus transformation and feruloyl esteraseproduction

Fungal cotransformation was carried out as describedby Punt and van den Hondel [21] using the faeB expres-sion vector and pAB4-1 [22] containing the pyrG selec-tion marker, in a 10:1 ratio. Transformants were selectedfor uridine prototrophy. Cotransformants containingexpression vectors were selected as described in the fol-lowing section. In addition, a control was transformedwith the pyrG gene but without the expression vector.

1 Abbreviations used: BRFM, Banque de Ressources Fongiques deMarseille; MCA, methyl caVeate; MpCA, methyl-p-coumarate; PAGE,polyacrylamide gel electrophoresis.

128 A. Levasseur et al. / Protein Expression and PuriWcation 37 (2004) 126–133

To screen the feruloyl esterase production in liquidmedium, 100 mL of culture medium containing 70 mMNaNO3, 7 mM KCl, 200 mM Na2HPO4, 2 mM MgSO4,glucose 5% (w/v), and trace elements was inoculated by1 £ 106 spores mL¡1 in a 500 mL baZed Xask. The cul-ture was monitored for 12 days at 30 °C in a shaker incu-bator (130 rpm). pH was adjusted to 5.0 daily with a 1 Mcitric acid solution. For protein puriWcation, 730 mL cul-tures were prepared in the same conditions. Each culturecondition was performed in duplicate.

Screening of the feruloyl esterase activity

Transformants were plated on a selective minimalmedium (without uridine) and incubated for 8 days at30 °C. To screen transformants, 40 individual cloneswere cultivated and checked daily for feruloyl esteraseactivity.

From liquid culture medium, aliquots (1 mL) werecollected and mycelia were removed by Wltration.Esterase activity was assayed as previously described[23] using methyl-p-coumarate (MpCA) or methyl caVe-ate (MCA) as substrate. Activities were expressed innkatal (nkat), 1 nkat being deWned as the amount ofenzyme that catalyses the release of 1 nmol of p-couma-ric or caVeic acids per second under establishedconditions.

Each experiment was done in duplicate and measure-ments in triplicated, and the standard deviation was lessthan 1% of the mean.

PuriWcation of the recombinant feruloyl esterase B

To purify the recombinant FAEB from A. niger,730 mL of a 8-day-old culture (8.7 nkat mL¡1 againstMCA) was Wltered (0.7 �m) and concentrated 14.6-foldby ultraWltration through a cellulose PLGC membrane(molecular mass cut-oV of 30 kDa) (Millipore, Saint-Quentin Yvelines, France).

The medium was dialysed overnight at 4 °C against a30 mM Tris–HCl, pH 7.0, binding buVer, loaded onto aChelating Sepharose Fast Flow column (13 £ 15 cm)(Amersham Biosciences, Orsay, France) previouslycharged with 0.2 M NiSO4 solution, and equilibratedwith Wve column volumes of binding buVer. After exten-sive wash with binding buVer, bound proteins were theneluted with 2 column volumes of an imidazole gradient(0–130 mM) in binding buVer at a Xow rate of1 mL min¡1 and collected with fractions of 8 mL.

Characterization of the recombinant feruloyl esterase B

Protein analysisProtein concentration was determined according to

Lowry et al. [24] with bovine serum albumin as standard.Protein puriWcation was followed by SDS–polyacrylamide

gel electrophoresis on 10% polyacrylamide slab gels [25].Proteins were stained with silver nitrate. Analytical iso-electric focusing was performed with 3–9 precast gel(Amersham Biosciences, Saclay, France) using a PhastSystem equipment according to the manufacturer’s pro-cedure. Molecular mass of the enzyme was estimated bygel Wltration on a Superdex 200 10/300 GL column(Amersham Biosciences, Saclay, France) with gel Wltra-tion kit, high molecular mass (Amersham Biosciences) asstandards.

N-terminal amino acid sequence determinationThe N-terminal sequence was determined according

to Edman degradation from an electroblotted FAEBsample (90 �g) onto a polyvinylidine diXuoride mem-brane (Millipore, Saint-Quentin Yvelines, France).Analyses were carried out on an Applied Biosystem470A.

Temperature stabilityPuriWed recombinant FAEB were incubated at vari-

ous temperatures (40–60 °C) for diVerent times. Aftercooling at 0 °C, FAEB activity was assayed in standardconditions against MCA.

Temperature and pH optimaFeruloyl esterase activity of the puriWed recombinant

FAEB was assayed in standard conditions at the variousdesigned temperatures from 40 to 60 °C against MCA assubstrate. For determination of the pH optimum, FAEBactivity was assayed in 100 mM citrate–phosphate buVer(pH 5.5–7.0) at 0.5 pH increments.

Results

Cloning of the faeB gene

The faeB gene was ampliWed from A. nigerBRFM131 genomic DNA using two speciWc primersdesigned from the faeB sequence (GenBank AccessionNo. AJ309807). The upstream primer conserved the sig-nal sequence of the faeB gene required for the endoplas-mic reticulum translocation. The downstream primerintroduced a six histidine codon to allow an eYcient tagfor subsequent puriWcation. Compared to the alreadypublished results [11], sequencing of the resultant PCRproduct revealed a DNA and amino acid sequence iden-tity of 92.8 and 97.5%, respectively, conWrming that weisolated the faeB gene from the strain A. nigerBRFM131. The amino acid (aa) changed for FAEB ofstrain BRFM131 are as follows: 21, P to S; aa 24, S toA; aa 28, E to D; aa 60, V to T; aa 132, G to A; aa 239,A to G; aa 240, N to D; aa 255, L to P; aa 285, G to S;aa 324, S to A; aa 326, S to Y; aa 486, N to D; and aa505, I to V.

A. Levasseur et al. / Protein Expression and PuriWcation 37 (2004) 126–133 129

Transformation and screening

In a cotransformation experiment, A. niger D15#26was transformed with a vector containing the faeBsequence and a pAB4.1 vector containing auxotrophicselection marker. Transformants were selected for theirabilities to grow on a minimum medium without uridineand were inoculated in liquid medium. Forty clones werecultivated in 50 g L¡1 glucose liquid medium and assayeddaily for esterase activity. Esterase activity ranged from2.3 to 18 nkat mL¡1 against MCA. The best clone wasselected in order to study the time course of the feruloylesterase activity.

Study of the recombinant feruloyl esterase B production

Esterase activity against MCA was detected on day 3and increased strongly until day 11 to reach a maximumof 18 nkat mL¡1. Then esterase activity decreased proba-bly due to proteolytic degradation (Fig. 1). Electropho-retic mobility of the total secreted proteins in the culturemedium was checked on SDS–polyacrylamide gel(Fig. 2). A predominant band around 75 kDa wasobserved corresponding approximately to the estimatedmolecular mass of the FAEB monomer.

PuriWcation and characterization of the recombinantFAEB

PuriWcation procedureRecombinant FAEB was puriWed from the culture

medium of A. niger by a one-step chromatographicstrategy (Table 1). The expression cassette for homolo-gous production of recombinant FAEB was designed toproduce a histidine tag at carboxy terminal end of therecombinant protein. Culture liquid medium was

Fig. 1. Feruloyl esterase (FAEB) production in the recombinant A.niger. Activity was measured using MCA as substrate.

harvested after only 8 days of growth to avoid proteo-lytic degradation generally occurring at the end of theculture and concentrated 14.6-fold by ultraWltration witha recovery of 82%. Total dialysed proteins were loadedonto a Chelating Sepharose Fast Flow column previ-ously charged with nickel. After extensive washing,bound proteins were eluted by competitive interactionwith imidazole. This sole aYnity chromatographic stepallows one to obtain 22.4 mg of puriWed protein with ahigh recovery of 64% and a puriWcation factor of 14.

Molecular mass and isoelectric pointThe homogeneity of the recombinant FAEB was

checked by SDS–PAGE showing a single band corre-sponding to the puriWed recombinant enzyme (Fig. 2).The molecular mass was estimated to 75 kDa in goodagreement with the FAEB from A. niger [11]. Moreover,a molecular mass around 150 kDa was estimated fromthe elution of the native recombinant FAEB on gelWltration chromatography, conWrming that the protein isa homodimer as the corresponding native FAEB. Con-cerning isoelectric point, at least four bands, correspond-ing to acidic proteins, were visible on the gel with amajor band at a pI of 4.8. This result might be explainedby the presence of several isoforms with diVerence ofglycosylation.

N-terminal sequencingTo conWrm that the puriWed recombinant protein is

the A. niger FAEB and that the maturation of the pro-tein is correct, the Wrst 10 amino acids were sequenced(ATDSFQARCN). In addition, alignment of N-terminalrecombinant FAEB with sequence deduced from thefaeB gene [11] reveals 80% identity and 90% similarity.

Fig. 2. SDS–PAGE analysis of the recombinant FAEB. The total pro-teins (lane 1) and puriWed FAEB 24 �g (lane 2) were loaded onto aSDS–PAGE (10%). The gel was silver stained. SD molecular weightmarkers.

130 A. Levasseur et al. / Protein Expression and PuriWcation 37 (2004) 126–133

EVect of temperature and pH on recombinant FAEBactivity

Using methyl caVeate as substrate, temperature andpH optima were 50 °C and 6.0, respectively.

Temperature stability of the recombinant FAEBTo evaluate the potential utilization of the FAEB, we

studied the enzyme stability, which was not determineduntil now, performing diVerent temperatures of preincu-bation from 40 to 60 °C (Fig. 3). The enzyme is stable at40 °C and its activity decreases by less than 10–16% after120 min at 45 and 50 °C, respectively. At 55 °C, the half-life of the enzyme was estimated to 30 min while at 60 °C,the recombinant FAEB was rapidly inactivated.

Kinetic propertiesTable 2 presents the kinetic constants (Km, kcat, Vmax,

and kcat/Km) calculated from the initial rate activity of

Fig. 3. Activity of the puriWed recombinant FAEB was assayed afterincubation at various temperatures using MCA as substrate. Selectedtemperatures were 40 °C (�), 45 °C (�), 50 °C (�), 55 °C (�), and60 °C (�).

Table 2Kinetic properties of the recombinant FAEB

Kinetic values were determined using methyl esters: methyl caVeicacid, methyl-p-coumaric acid, and chlorogenic acid (CA) as substratesand determined from a Lineweaver–Burk plot.

Kinetic constants

Km (mM)

kcat

(kat mol¡1)Vmax (nkat mg¡1)

kcat/Km (M¡1 s¡1)

MpCA 0.029 77 527.6 2.65 £ 106

MCA 0.134 161 1101 1.2 £ 106

CA 0.16 4.1 28.3 2.56 £ 104

recombinant FAEB against MCA and MpCA. Km andVmax were determined from Lineweaver–Burk plot anal-ysis. Using methyl caVeate and methyl-p-coumarate assubstrates, the Km values were 0.13 and 0.029 mM,respectively. kcat values for recombinant FAEB variedtwofold between MpCA and MCA (77 and161 kat mol¡1). Catalytic eYciency was greater forMpCA than MCA 2.65 £ 106 and 1.2 £ 106 M¡1 s¡1,respectively. Activity of the recombinant FAEB was alsomeasured using chlorogenic acid (5-O-caVeoyl quinicacid) as substrate (an ester between caVeic acid and qui-nic acid). Km value (0.16 mM) was of the same order asmethyl caVeate (0.134 mM) but kcat was signiWcantlylower (40-fold) indicating the poor eYciency of FAEBfor chlorogenic acid.

Discussion

Feruloyl esterases are essential accessory enzymesacting in synergy with main-chain degrading carbohyd-rases to allow an eYcient degradation of plant cell.Potential applications of esterases in the food and cos-metic industries and in the pulp and paper sectorincrease interest for this enzyme group [27]. To obtainhigh quantities of FAEB that were not available fromthe wild-type strain, we used an homologous overexpres-sion process with A. niger as host strain. This organism isa well-known host for protein overproduction [28,29]and allows the utilization of the recombinant enzyme infood process as the species obtained a GRAS (generallyregarded as safe) status [30]. In this study, the expressioncassette contains the genomic DNA encoding theA. niger FAEB with its own signal sequence to driveFAEB secretion. The gene was cloned from A. nigerBRFM131 and compared to the previously publishedfaeB gene obtained from A. niger CMICC92980. Align-ment of both genomic sequences and deduced aminoacid sequences showed a 92.8 and 97.5% identity, respec-tively. In addition, N-terminal sequence of the recombi-nant FAEB was demonstrated to present 80% identitywith the native FAEB. The diVerences could beexplained by intraspecies variations. These results couldprovide some insights into evolutionary forces that con-straint FAEB sequence, suggesting that modiWed aminoacids must have a weak functional constraint as recom-binant and native FAEB was demonstrated to present

Table 1PuriWcation of the recombinant FAEB from A. niger

Activity was measured using MCA as substrate.

PuriWcation steps Protein (mg)

Total activity (nkat)

SpeciWc activity (nkat mg¡1)

Activity yield (%)

PuriWcation factor

1. Culture medium 489 6380 13 100 12. UltraWltration 115 5245 45.6 82 3.53. IMAC 22.4 4105 183 64 14

A. Levasseur et al. / Protein Expression and PuriWcation 37 (2004) 126–133 131

the same physico-chemical properties. These resultsshould be further discussed when the structure will beresolved.

From transformants screening procedure using ester-ase activity assay, we isolated a clone that produces after11 days of growth around 100 mg L¡1 based on the spe-ciWc activity of the FAEB using MCA as substrate.FAEB production was estimated to be 16-fold higher ascompared to the production level obtained with thewild-type strain BRFM131 under optimal conditionsusing sugar beet pulp as inducer. This improvement fac-tor is in good agreement with homologous overproduc-tion, generally ranged from 5 to 50. For instance, thehomologous overproductions of catalase and a majorendopolygalacturonase from A. niger allow one toobtain an improvement factor of 10 and 50, respectively[31,32]. Recently, homologous overproduction of FAEAfrom A. niger was successfully achieved with improve-ment factor of 50-fold and production yield estimated at1 g L¡1 [19]. FAEA was equally produced in Pichia pas-toris as host organism but with a lower secretion yield upto 300 mg L¡1 [33]. In addition, two type B esterases pro-ductions were performed in the past. FAEB gene fromPenicillium funiculosum was expressed in Escherichia colicells as fusion with the GST protein but the resultingFAEB-GST fusion was produced as insoluble inclusionbodies leading to a weak yield of feruloyl esterase activ-ity after denaturation and refolding of the recombinantenzyme [26]. A type B feruloyl esterase from Neurosporacrassa was successfully overproduced in P. pastoris witha secretion yield around 210 mg L¡1 but no comparisonof characteristics between both recombinant and nativeproteins was performed [34]. However, the previousFAEA homologous overproduction was found to be 10-fold more eYcient as compared to the FAEB produc-tion. This tendency was observed for the wild-typestrains under induction conditions [8]. Weaker yield ofoverproduction for FAEB as compared to the FAEAusing the same host strain might have arisen from threemajor reasons. First, the number of copies and locationof the vector integration could be a determinant factordirectly linked to the expression level of the correspond-ing gene [32]. In the second place, the signal sequence ofFAEB could be limiting point that could markedly inXu-ence the production yield [29,31]. Indeed, signal peptidefrom well-secreted homologous protein is often fused tothe mature recombinant protein to facilitate its secretion[35]. Lastly, FAEB is a relatively high molecular massprotein synthetized as an homodimer and therefore, theglobal yield of production of this protein could beslowed down, as compared to a small monomeric pro-tein such as FAEA, because of post-translational pro-cessing constraints due to the size, the organization, andthe glycosylation extent of the protein [36].

The secreted recombinant FAEB from a 8-day-oldculture was puriWed to homogeneity by a one-step puriW-

cation procedure compatible with large-scale industrialproduction. The global recovery of 64% was close to thatobtained for the previous puriWcation of the recombi-nant FAEA, i.e., 69% [19]. However, puriWcation factorwas higher, i.e., 14 instead of 3.5 because of contaminantproteins that were more represented in the present pro-duction. The main physico-chemical properties of therecombinant FAEB were determined to be compared tothose of the native FAEB [8]. All main characteristicspreviously determined for the recombinant FAEB, i.e.,molecular mass (75 kDa), isoelectric point (4.8), temper-ature (50 °C), and pH (6.0) optima were in completeagreement with characteristics of the native FAEB [8,11].In the present work, isoelectric focusing analysisrevealed several acidic isoforms, presumably related tovarious glycoforms of recombinant FAEB probably dueto the 18 potential glycosylation sites. These diVerentisoforms were not shown previously for native FAEB.However, presence of type B feruloyl esterase isoformswas only observed for FAEB from P. funiculosum. Asour best transformant was selected for its high level ofFAEB production, the glycosylation pattern might diVerfrom that of the wild-type strain simply due to theincreased Xux of FAEB through the endoplasmic reticu-lum and Golgi apparatus. A limited chaperone and fold-ases level as compared to the high production ofrecombinant FAEB could prevent a correct processingof FAEB as glycosylation. Therefore, some steps duringoligosaccharide processing may have been omitted ortheir regulation altered [36–38].

Applications of esterases in the pulp bleaching treat-ment required high temperature incubation [19]. Toappreciate the potential utilization of recombinantFAEB in this sector, the temperature stability was stud-ied as there were no data concerning the native proteinusing MCA as substrate. The commonly temperatureused for pulp treatments was around 50 °C. At this tem-perature, recombinant FAEB activity decreases by lessthan 10–15% after 2 h incubation. The temperaturestability is in complete agreement with an enzymatictreatment in this application procedure.

The kinetic constants of the recombinant FAEB forsynthetic methyl ester of hydroxycinnamic acids weremeasured and compared with those of the nativeenzyme. As previously described kcat values of therecombinant FAEB are higher with MCA than MpCA(161 and 77 s¡1, respectively) but Km for MpCA is signiW-cantly lower than with MCA. As a consequence, the cat-alytic eYciency (kcat/Km), which is a good indicator ofenzyme eYciency and speciWcity, is better for MpCAthan MCA. This is in agreement with previouslyreported values for A. niger FAEB but also for P. funicu-losum and N. crassa [26,34]. SpeciWty of FAEB for anatural substrate was also tested with chlorogenic acid(5-O-caVeoyl quinic acid). This latter is present as solu-ble component found in vegetables and fruits [39]. When

132 A. Levasseur et al. / Protein Expression and PuriWcation 37 (2004) 126–133

the activity was measured with chlorogenic acid andcompared with that of methyl caVeate, a similar Kmvalue was obtained (0.16 and 0.13 mM) but kcat was40-fold lower leading to a dramatic decrease of thecatalytic eYciency. It was already shown that feruloylesterase activities were greatly inXuenced by the substi-tutions on the benzene ring, the distance betweenaromatic ring and the ester bond. These results conWrmthe determining role of the molecules adjacent to theester bond for the correct alignment of the substrate inthe active site.

As a conclusion, we fully succeed for the Wrst time inthe homologous overexpression of the faeB gene inA. niger. The main characteristics of the recombinantFAEB were in good agreement with those of the corre-sponding native protein and new insights were providedin order to present a more complete description of thisenzyme. SuYcient amount of proteins obtained from theFAEB overproduction will allow structure–functionstudies to be carried out. In addition, these promisingresults of overproduction will permit us to envisage theWrst experiments of application and to check the potenti-ality of this enzyme in pulp and paper or more generallyin biotransformation of phenolic compounds of agricul-tural by-products.

Acknowledgments

This research was supported by the GIS-EBL (Cons-eil Régional Provence-Alpes–Côte d’Azur, the ConseilGénéral 13, France) and by grants from the MENRT(Ministère de l’Education Nationale, de la Rechercheet de la Technologie, France) and TEMBEC S.A. com-pany. We thank Dr. P.J. Punt and Prof. C.A.M.J.J. vanden Hondel (TNO, Zeist, The Netherlands) for provid-ing plasmid PAN523 and A. niger D15 as fungal host forthis study.

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