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Biochimica et Biophysica Act

Laccase of Cyathus bulleri: structural, catalytic characterization andexpression in Escherichia coli

Salony, N. Garg, R. Baranwal, M. Chhabra, S. Mishra ⁎, T.K. Chaudhuri, V.S. Bisaria

Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi 110016, India

Received 10 February 2007; received in revised form 18 October 2007; accepted 5 November 2007Available online 22 November 2007

Abstract

Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several whiterot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion ofsome basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% α-helical, 26% β-sheet and38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher β-sheet structures.The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and thiswas confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme wasdenatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments underalkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. Thelaccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to beexpressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.© 2007 Elsevier B.V. All rights reserved.

Keywords: Laccase; Cyathus bulleri; Circular dichroism spectroscopy; Laccase expression

1. Introduction

Lignin is a highly cross-linked aromatic polymer formedfrom monomeric units of phenylpropanoid compounds. It actsas a structural adhesive, holding cellulose microfibrils togetherin the plant cell wall. Since the degradation of lignin is a majorobstacle to efficient utilization of lignocellulosic materials [1]considerable work has been done in the area of microbial lignin-degrading enzymes. The white rot fungi, which belong toBasidiomycetes, are the only organisms capable of selectivelydegrading lignin to carbon dioxide and water thereby increasingthe forage digestibility and adding to its protein content [2,3].These fungi secrete extracellular enzymes like lignin perox-idase, managanese peroxidase and laccases, which are respon-sible for degradation of lignin. Laccases can oxidize phenolic

⁎ Corresponding author. Fax: +91 11 2658 2282.E-mail addresses: saroj@dbeb.iitd.ernet.in, saroj98@hotmail.com

(S. Mishra).

1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbapap.2007.11.006

compounds, thereby creating phenoxy radicals, while non-phenolic compounds are oxidized via participation of cationradicals in laccase mediated systems. Some white rot fungicontain all the three classes of the lignin modifying enzymeswhile others contain one or two classes of these enzymes [4].

Laccases (bezenediol: oxygen oxidoreductases [EC 1.10.3.2])are copper containing enzymes which catalyze the oxidation ofa broad range of phenolic compounds and aromatic amines byusing molecular oxygen as the electron acceptor. They containfour Cu (II) ions arranged in three different sites. The reactionmechanism proposed for these enzymes is supported by theelectron transfer reactions that occur between cupric ions duringcatalysis [5]. Laccases have become industrially important dueto their potential use in diverse applications which includewaste detoxification, textile dye transformation, personal andmedical care, biosensor and analytical applications [6]. Forsome laccases, the substrate specificities have been extended tonon-phenolic subunits of lignin, various dyes, polyaromatichydrocarbons and polychlorinated biphenyls by using redoxmediators [7].

260 Salony et al. / Biochimica et Biophysica Acta 1784 (2008) 259–268

Laccases are encoded by complex families of structurallyrelated genes. At least 29 fungal genes have been cloned whichinclude five from Trametes villosa [8,9] and Trametessanguinea [10], four from Rhizoctonia solani [11], three fromBasidiomycetes I-62 (CECT 20917) [12], two from Agaricusbisporus [13], Pycnoporus cinnabarinus [14,15], Pleurotusostreatus [16] and one each from Neurospora crassa [17],Coriolus hirsutus [18], Phlebia radiata [19], Coprinuscongregatus [20],Mauginiella sp [21] and Volvariella volvacea[22]. Overall sequence identity between fungal laccases may below, but conservation is high within regions involved in copper-binding. Various fungal laccases have been expressed inheterologous eukaryotic hosts such as Aspergillus oryzae[9,23], Pichia pastoris [24–27], Saccharomyces cerevisiae[28,29] and Trichoderma reesei [30]. Clearly, the expression oflaccases in eukaryotes also raises the question of alternativeglycosylation patterns coupled with the difficulty of large-scalecultivation of many of these hosts, particularly the filamentousfungi. It would be highly advantageous to express the laccase inthe bacterium Escherichia coli, for which a large number ofexpression vectors are available and also cultivation strategiesfor achieving high cell density fermentations.

The bird's nest fungus, Cyathus bulleri, colonizes deadherbaceous stems, wood chips, dung, sticks and other woodydebris and has been found to be ecologically suitable for lignindegradation [31]. We [32] and another group [33] have recentlyreported on the purification of laccase from this fungus. Theenzyme exhibited some interesting catalytic properties and hasbeen used for decolourization of a number of reactive azo andnon-azo dyes [32,34]. In this paper, we describe somestructural and important catalytic properties of this enzyme.We also report on the expression of C. bulleri laccase in E. coliusing a modification of the ligation-anchored (LA)-PCRapproach [35]. The expression was confirmed by zymogramanalysis making it the first fungal laccase to be expressed inthis bacterium.

2. Materials and methods

2.1. Organism and culture conditions

C. bulleri (Brodie) 195062 obtained from Canadian Type Culture Collectionwas a kind gift from Dr. R. C. Kuhad (University of Delhi, South Campus). Thestrain was maintained at 26 °C on malt extract medium with the pH set to 5.2.For laccase production, the fungus was cultivated in basal liquid (BL) medium[36] with 2,6-dimethylaniline as inducer as described previously [32].

2.2. Purification of laccase

Routine assay of laccase was carried out using 10 mM guaiacol as substratein 50 mM phosphate citrate buffer, pH 5.0 as described [37]. The enzyme waspurified by 82-fold to a high specific activity of about 4000 U/mg protein asdescribed earlier [32]. The purified enzyme was stored at 4 °C and used for thestudies described in this paper.

2.3. Amino acid composition and comparison with generic laccases

Samples on PVDF membrane [38] were analysed for total amino acidcomposition at the Protein Chemistry Core Laboratory at Baylor College ofMedicine, Houston, Texas. The results reported are the average of three runs.

The amino acid composition of several laccases was computed in mol percentfrom the sequences downloaded from www.expasy.org. Only full lengthlaccases were used in the study. The SWISS PROT ID of the proteins analysedin this study is listed at the end in Appendix-I.

2.4. Far-UV CD spectrum of laccase

The Far-UV CD spectrum of purified laccase was determined in 10 mMsodium cacodylate buffer, pH 5.0 in a Jasco J-810 spectropolarimeter (JascoCorporation, Japan). For this, the enzyme was extensively dialyzed in this bufferat a protein concentration of 0.5 mg/ml. The instrument parameters used for themeasurement were: sensitivity—100 mdeg, wavelength—200 to 250 nm, datapitch—0.2 nm in continuous scanning mode. The scan speed was set at 50 nm/min with a response of 1 s and band width at 1 nm as described previously [39].The CD spectrum obtained from the instrument had the units in mdeg whichwere converted to molar residue ellipticity by using the molecular weight andconcentration of protein solution with the help of software associated with theinstrument. Secondary structural elements were calculated from the experi-mental CD values in deg cm2 dmol−1 using the software K2D [40].

2.5. Influence of pH and group specific inhibitors on laccase activity

Activity and kinetic parameters of purified laccase were determined using2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as the substrate[36].The purified laccase (5 μM) was incubated with different ABTSconcentrations (0.01–10 mM) in 20 mM glycine–HCl buffer (pH 2.0–3.0) or50 mM citrate buffer (pH 4.0–6.0). The apparent maximum velocity (Vmax)and Km were determined from the double-reciprocal plot. The higher and thelower pKa values were found graphically from the plot of logVmax againstpH [41].

The effect of various chelating, respiratory inhibitors and sulfhydral agents[EDTA (0.1–1 mM), sodium azide (0.005–0.05 mM), kojic acid (0.5–10 mM),DTT (0.01–1 mM), l-cysteine (0.05 and 0.1 mM) and p-coumaric acid (1 and5 mM)] was studied with ABTS as the substrate. The different group specificchemical modifiers like 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) and diethyl pyrocarbonate (DEPC) were also used to investigate thechemical nature of the amino acids involved in enzyme activity. Based onpromising lead with EDC and the sharp decline in enzyme activity at pHvalues greater than 4.5, this inhibition was studied in detail. The purifiedenzyme (1.5 μM) was incubated with different concentrations of EDC (50–200 mM) at 25 °C as described [42]. Aliquots of 50 μl were withdrawn atregular time intervals and added to 50 μl of 50 mM citrate buffer, pH 6.0 toquench the reaction. The residual enzyme activity was measured usingguaiacol as substrate and expressed as the percentage of unmodified control.The log of percentage residual activity was plotted against time for differentEDC concentrations.

Pseudo-first order rate constants (kapp) were determined from the slopes ofsemi-logarithmic plots of residual activity against time for different EDCconcentrations. The second-order rate constant was determined from the plot ofkapp vs. EDC concentration. The order of the reaction (n) was determined asdescribed [43]:

logkapp ¼ logk þ n log I½ �where k is the second-order rate constant and [I], the inhibitor concentration.

2.6. Unfolding and refolding of purified laccase in the presence ofCu2+ and Zn2+ ions

For unfolding of the purified laccase, chemical reagents like EDTA, DTTand guanidinium hydrochloride (GdnHCl) were used at different concentrations(EDTA: 1–100 mM, DTT: 50–200 μM, GdnHCl: 1–6 M). The conditions i.e.time and concentration, were optimized for complete loss of activity at theminimum inhibitor concentration with 0.1 mg of protein in a reaction volume ofone ml. The unfolded protein was refolded using alkaline conditions for 30 minfollowed by addition of 1 mM copper or zinc. Aliquots were removed at regularshort intervals of time and activity of the enzyme monitored using guaiacol asthe substrate.

Table 1Oligonucleotides used in the study

Oligonucleotide Sequence

LCC-CS 5′-GGG TTG TCG TAG TTG TA-3′LCC-CD 5′-GGR TTR TCR TAR TTR TA-3′LCC-CDT 5′-CCC TCA NGG RTT RTC RTA RTT RTA-3′LCC-NS 5′-CCN GAT GGN TTR CCC G-3′LCC-ND 5′-CCN GAY GGATTR CCA G-3′Anchor 5′-T CCC TTT AGT TGA GGG TTA ATA TAA GCG

GCC GCG TCG TGA CTG GGA GCG C-3′P 2 (Complementaryto anchor)

5′-ATT AAC CCT CAA CTA AAG GGA-3′

P 3 5′-ATC AAC TCG GCYATC CT 3′P4 5′-SGG GTT GTC GTA GTT GTA 3′

Where R=A/G, N=A/C/G/T, Y=C/T, S=C/G.

261Salony et al. / Biochimica et Biophysica Acta 1784 (2008) 259–268

2.7. RNA isolation and enrichment of poly A+RNA

The fungus was grown in BL medium under static conditions as describedabove. On day 5 of growth, 100 μM of 2,6-dimethylaniline was added andmycelium were harvested after 24 h. Total RNA was isolated in a single-stepmethod using acid guanidinium thiocyanate-phenol-chloroform extractionmethod [44]. RNA was enriched for polyA mRNA using the Qiagen kit. ThemRNA preparation was visualized using denaturing gel electrophoresis to verifythe quality of the prep.

2.8. Cloning and expression of laccase in E. coli

The strategy of cloning is described in Fig. 1 and was a modification of theLA-PCR method [35]. Briefly, the mRNA was transcribed to cDNA by usingSuperscript reverse transcriptase II (Life technologies) using oligo dT as aprimer according to the instructions. The 3′-end of the reverse transcribed singlestranded cDNAwas ligated to a 5′-phosphorylated (with polynucleotide kinase),3′-end blocked (with ddATP using terminal deoxynucleotidyltransferase) anchoroligonucleotide (5′ T CCC TTT AGT TGA GGG TTA ATA TAA GCG GCCGCG TCG TGA CTG GGA GCG C 3′) using T4 RNA ligase as described [35].The resultant anchored product was subjected to PCR amplification using theforward primer P 2 (5′ ATTAAC CCT CAA CTA AAG GGA 3′), based on thesequence within the anchor, or, LCC-NS/LCC-ND, based on the published [32]peptide 2 sequence, PDGFP. This peptide was hypothesized (based on

Fig. 1. Protocol for modified LA-PCR approach. First strand cDNA synthesiswas performed with avian myeloblastosis virus reverse transcriptase and oligo(dT) as primer [35]. The 5′-phosphorylated, 3′ blocked anchor oligonucleotidewas ligated to first strand cDNA. Two primers, P2, specific to a sequence withinthe anchor and the second reverse primer was designed based on one of theinternal peptide sequences [32] of purified laccase. The reverse primer had eithera termination codon or did not have a termination codon as described in the text.The PCR products (solid box) were independently cloned into PCR 2.1 vector(Invitrogen).

comparison with other published laccase protein sequences) to be towards theN-terminus of the purified laccase of C. bulleri. These two forward primers wereused for amplification of laccase specific gene in conjunction with a number ofreverse primers. These were (LCC-CS, LCC-CD, LCC-CDT) and weredesigned based on the internal peptide sequence 1 (YNYDNP) of the purifiedC. bulleri laccase [32].This sequence was proposed to be towards the C-terminus of the laccase, based on comparison with published laccase sequences.The sequences of these reverse primers are listed in Table 1. Within the reverseprimer either a termination codon was introduced (LCC-CDT) or not introduced(LCC-CD) leaving the product that would be translated, to run into a terminationcodon naturally on the plasmid vector. The PCR was performed with 2 μlaliquots of the terminated ligation mixture in 50 μl reaction volume containingTaq polymerase (Perkin Elmer). The reactions were run using thermal cycler(Perkin Elmer) with a temperature program of: initial denaturation at 94 °C for5 min, followed by 30 cycles of initial denaturation at 94 °C for 1 min, annealingat 48 °C for 1 min and extension at 72 °C for 1 min and a final extension at 72 °Cfor 10 min. Appropriate changes in annealing temperatures were madedepending on the primer sequences. The resultant products were run on 1%agarose gel to confirm nature of amplifications. The PCR reaction products,where amplification of high molecular weight DNA had occurred (Fig. 5, lane2), were purified by Clean Genei Kit (Bangalore Genei Pvt. Ltd., Bangalore) andligated to pCR 2.1 vector that has 3′ T overhangs. The ligation mixture wastransformed into One Shot cells (INVαF′, TOP 10 F′) (Invitrogen) as permanufacturer's instructions. The transformants were plated on LB agar platecontaining X-Gal and 50 μg/ml kanamycin and 50 μg/ml ampicillin. The stablewhite transformants were screened for the laccase gene by colony hybridizationusing the same degenerate primer (LCC-CD) that was used for amplification.The conditions for preparation of transformants for colony hybridization were asper standard protocols [45].

2.9. Labelling of oligonucleotide and hybridization

The oligonucleotide (LCC-CD) was labeled with [γ-32P] ATP (BRIT,Hyderabad) using the enzyme T4 polynucleotide kinase. The reaction mixturecontained the oligonucleotide (50 pmol), 2 μl of 10× bacteriophage T4polynucleotide kinase buffer, 5 μl of [γ-32P] ATP, 11.4 μl of sterile water and1 μl (10 U) of the enzyme. The efficiency of the transfer of γ-32P to theoligonucleotide was checked by measuring the amount of radioactivity using ascintillation counter (Kontron, Switzerland). The pre-hybridization andhybridization (∼5000 cpm) was carried out at 50 °C. The hybridized andwashed membrane was exposed to X-ray film (X-OMAT, Kodak) and incubatedat −70 °C overnight. The film was developed in an automatic X-ray developer(Kodak).

2.10. Screening of positive recombinant clones by expression

Single colonies of the transformants which gave a positive signal with theprobe were grown in LB+ampicillin (50 μg/ml) at 37 °C overnight. This wasused to start a fresh day-culture in a 25 ml flask. At OD600 of 0.5–0.6, IPTG(final concentration 1 mM) was added for induction. The cells were harvested by

Table 2Comparison of mol % amino acid composition of Cyathus bulleri laccase withother known generic laccases (Swiss PROT ID of laccases is provided inAppendix-I)

Amino acid Mol% of generic laccases Mol% of C.bulleri⁎

Ala (A) 7.73±1.02 8.37Cys (C) 1.10±0.09 –Asp (D)+Asp (N) 8.88±1.16 10.17

7.48±0.76Glu(E)+Gln (Q) 2.68±0.78 11.27

4.40±0.93Phe (F) 8.58±1.11 3.28Gly (G) 5.51±0.48 17.76 a

His (H) 4.97±0.58 4.75 b

Iso (I) 6.71±0.79 4.32Lys (K) 1.82±0.60 4.06Leu (L) 9.83±0.91 7.22Meth (M) 1.37±0.34 –Pro (P) 8.34±0.83 4.29

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centrifugation at 5000 g at regular intervals. The supernantant was collectedseparately and the cell pellet was washed with phosphate buffered saline, pH 7.2.The cell pellet was suspended in 50 μl SDS-PAGE buffer and directly loaded(without boiling) on 10% SDS-PAGE. The gel was electrophoresed at 4 °C.Activity was detected by zymogram staining (100 ml of staining solutioncontained 60 mg diaminobenzidine and 30 mg NiCl2.6H2O in 50 mM sodiumacetate buffer, pH 4.5) as described earlier [32]. A control host with only thevector and a preparation of the purified C. bulleri laccase (∼8 μg protein) wererun on the same gel for comparison.

2.11. Determination of the partial sequence of the laccase gene

An aliquot (5 μl) each of the reverse transcribed mRNA and PCR amplifiedproduct obtained using P2/LCC-CD (Fig. 5, lane2) was again used in a new PCRreaction set up in a total volume of 25 μl. The primers (Takara, HPLC purified)used were:

(corresponding to thepublished [32] internal

Arg (R) 5.17±0.79 3.85Ser (S) 7.06±0.86 9.82Thr (T) 8.69±1.28 6.04Val (V) 8.81±0.94 4.96Trp (W) 2.60±0.28 not determinedTyr(Y) 4.89±0.55 2.08

⁎Values represented are averages for 3 replicates. (–) = Hydrolysed and cannotbe determined from this analysis.a

peptide sequence, G(?)reported earlier was notused in designing theprimer as in most lac-cases the analogousamino acid isN)

(the complementarysequence of which is

Most likely due to transfer conditions in Tris–glycine buffer.b The value is an estimate, as it could not be quantitated accurately due to

closeness to Gly peak.

given below)

(corresponding to the pub-lished internal peptide seq-uence of C. bulleri laccase[32]).

The reaction vial contained 0.2 mM of each dNTP, 50 pmol each of P3 andP4 and 1.25 U of Takara Ex Taq™ (Takara Bio Inc., Japan). The conditions forPCR amplification were initial denaturation at 94 °C for 7 min followed by35 cycles of 1 min denaturation at 94 °C, 1 min annealing at 48 °C and 2 minextension at 72 °C. The final extension was carried out for 10 min at 72 °C. A10 μl aliquot was run on the gel to confirm amplification of the product. Thefragment was eluted out of the gel using Qiagen gel cleaning kit andnucleotide sequence determined on an automated DNA sequencer (ABI prism ,Model 3730) at the Department of Biochemistry, Delhi University SouthCampus. Both P3 and P4 were used for sequencing of the fragment. Thededuced amino acid sequence was submitted to NCBI and % sequencesimilarity noted with the reported laccase sequences. The top 10 similarsequences were aligned with the Clustal V program [46] of DNA-Star (fordetails, see legend to Fig. 6). The sequence of the 450 bp fragment of laccase,reported in this paper, has been deposited in the GenBank database underAccession No. EU195884

3. Results

3.1. Amino acid composition of purified laccase andcomparison with generic fungal laccases

The amino acid composition of the purified laccase wascompared with the mol% values obtained for other fungallaccases (Table 2). The results indicated a high proportion(N21.44%) of Asp, Asn, Glu and Gln and 7.91% of basicamino acids Lys and Arg in the purified laccase. However, theC. bulleri enzyme showed lesser percentage of hydrophobic(Phe, Iso, Leu, Pro, Val, Tyr) amino acids. This was interesting

in view of the fact that very low standard deviation (0.02–1.28%) was obtained for the amino acids within the generallaccase category. The unusually high content of glycineobtained for the laccase of C. bulleri was possibly due totransfer conditions.

3.2. Far-UV CD spectrum of laccase

The Far-UV CD spectrum of the purified laccase is shownin Fig. 2. The CD spectrum showed two negative troughs,one around 220 nm and the other at 208 nm which are atypical signature for all α, all β or α+β proteins withdifferent intensities corresponding to the extent of secondarystructural elements [47]. For the a/β proteins, the trough atthe shorter wavelength is always skewed towards 220 nm andone broad negative trough is observed around 220 nm. Onthe other hand, proteins with no organized structure display anegative trough around 200 nm. The content of secondarystructure in the purified laccase was calculated from theexperimental CD values according to Yang et al. [40] basedon model peptides. It contained approximately 37% α-helix,26% β-sheet and 38% random coil. Based on theseobservations, the laccase was proposed to belong to α+βstructural class.

3.3. Effect of pH on laccase activity

The effect of pH on laccase activity was determined bystudying the kinetic parameters on ABTS. The range of pH

Fig. 3. Effect of pH on catalytic activity of purified laccase of C. bulleri. Theenzyme was incubated with six different substrate concentrations, prepared inbuffers ranging from pH 2.0 to 6.0 and Vmax was calculated at each pH from thedouble-reciprocal plots. The enzyme was stable in the pH range of 3.0–6.0.

Fig. 2. Far-UV CD spectrum of purified laccase in 10 mM sodium cacodylatebuffer pH 5.0 at 25 °C. Protein concentration was 0.5 mg/ml. CD spectrum wasmeasured in a quartz cuvette of 1 cm path length and the value of ellipticity wasconverted to molar ellipticity as described in the text.

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selected was 2.0–6.0. The value of Vmax was calculated fromthe plot of 1/V vs. 1/[s] at each pH values. The values of pKa1

and pKa2 were determined graphically as shown in Fig. 3 andfound to be 3.5 and 4.65 respectively. A sharp drop in activitywas seen at pH values lower than 3.0 and more than 4.65suggesting titration of important catalytic groups or enzymeinstability in these pH regions. Since the enzyme has beenreported to be stable up to pH 6.0, the decline in the activity atpHN4.5 appeared to be linked to ionization of an importantcarboxylic group (Asp/Glu).

The sensitivity of the purified laccase towards several rea-gents was studied. The inhibitor sodium azide (at 0.05 mM),Kojic acid (5 mM), reducing agents DTT (1 mM), andl-cysteine (0.1 mM) completely inhibited laccase activity.The metal chelators like p-coumaric acid and EDTA showedpartial inhibitions which was about 60% with 5 mM p-coumaric acid and 67% with 1 mM EDTA. There was noevident inhibition on the activity of laccase in the presenceof DEPC which couples His when it was tested in the rangeof 5 μM–50 mM. However, the group specific agent EDC,(at a concentration of 200 mM) nearly completely (80%)inhibited laccase activity. The kinetics of inhibition wasfollowed with EDC at various initial concentrations of theinhibitor (50–200 mM) and the results are shown in Fig.4A. EDC caused a time and concentration dependentdecrease in activity towards guaiacol. The semi-logarithmicplots of residual activity as a function of time were biphasic,indicating the complexity of EDC induced inactivation. Thepattern of this inhibition can be resolved into two first orderprocesses with slope of longer times determining the rate ofinactivation. The pseudo-first order rate constants were0.0038 min−1, 0.0176 min−1, 0.0185 min−1 and 0.025min−1 at 50 mM, 100 mM, 150 mM and 200 mM of EDCrespectively. The second-order rate constant of inactivationwas determined from a plot of pseudo-first order rate

constants as a function of EDC concentration. This wasdetermined to be 1×10−4 mM−1 min−1. Analysis of theorder of inactivation with respect to EDC concentrationyielded a slope of 1.32 (Fig. 4B) indicating that one moleculeof EDC binds to one molecule of enzyme when inactivationoccurred.

3.4. Unfolding and refolding of purified laccase

The purified laccase was subjected to unfolding usingEDTA, GdnHCl and DTT. The purified protein was incubatedwith the respective denaturant for one hour and laccase activitymeasured using guaiacol as the substrate. It was found thatEDTA at a concentration of 1 mM, GdnHCl at 4 M and DTT at100 μM resulted in loss of activity of the enzyme. The time fordenaturation with EDTA, DTT and GdnHCl was optimized tobe 1 hr. Since EDTA is a metal chelating agent, at highconcentration (1 mM), it was assumed that it bound to the metalions, resulting in the loss of enzyme activity. The denaturedprotein (with EDTA, DTT, GdnHCl and combination of thethree) diluted 100-fold with the citrate buffer (pH 5.5) did notshow much activity against guaiacol. Since laccase is a metallo-protein, the refolding experiment was done in the presence ofcopper. Addition of copper at pH 5.5 led to only 45% recoveryof the original activity. Longer times of incubation for refoldingresulted in precipitate formation. However, when refolding wasattempted at an alkaline pH of 8.0 for about 30 min and copperwas added at the end of the refolding period, almost 100%activity was recovered in 10 min after addition of copper. Therefolding was also carried out in the presence of zinc as theatomic radii of copper and zinc are similar. The refolding of thedenatured protein (with mixture of EDTA, DTT and GdnHCl)was done in citrate buffer (pH 5.5) and zinc was added after60 min. In this case, 100% activity was recovered in about5 min.

3.5. Cloning of laccase and its expression in E. coli

The LA-PCR method was used to clone the laccase gene.The results of the PCR experiment are shown in Fig. 5A. As

Fig. 4. (A) Kinetics of inhibition of purified laccase with EDC. The enzyme(1.5 μM) was incubated with 50 mM (♦), 100 mM (■), 150 mM (▴), or200 mM (■) of EDC at 25 °C. Aliquots of 50 μl were withdrawn after differentintervals of time (as indicated on x-axis) and added to citrate buffer, pH 6.0 toquench the reaction. Residual enzyme activity was measured against guaiacoland was expressed as the percentage of unmodified control (taken as 100%,log100=2). (B) A plot of log kapp (pseudo-first order rate constants for eachEDC concentration) as a function of EDC concentration to determine n (slope,which represents the order of the reaction) as explained in the text.

Fig. 5. (A) Analysis of LA-PCR products by 0.7 % agarose gel electrophoresis.Each PCR reaction used total cDNA as a template using different sets of primers.Lane 1: P2/LCC-CDT, Lane 2: P2/LCC-CD, Lane 3: P2/poly dT, Lane 4: P2/LCC-CDT, Lane 5: P2/LCC-ND, Lane 6:LCC-NS/poly dT, Lane 7:LCC-ND/poly dT, Lane 8: λ-DNA-EcoRI/HindIII cut. (B) PAGE-zymogram analysis ofthe whole cell extract of E. coli containing either the vector (lane 2) or theconstruct containing truncated version of the laccase gene of C. bulleri (lane 3).The purified laccase of the fungus was loaded in lane 1 as a positive control.Samples were solubilized in SDS solubilization buffer (but were not boiled) andloaded in 10% SDS-PAGE gel. Electrophoresis was performed at 4 °C. After therun, the gel was washed a few times in buffer and activity was detected byzymogram staining (100 ml of staining solution contained 60 mg diamino-benzidine and 30 mg NiCl2.6H2O in 50 mM sodium acetate buffer, pH 4.5). Thebands appeared within 10 min of incubation. The position of the mol wt standardmarkers (Bangalore Genei) is shown on the right, Myosin, Rabbit Muscle:205 kDa, Phosphorylase b: 97.4 kDa, Bovine Serum Albumin: 66 kDa,Ovalbumin: 43 kDa.

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seen, using P2 and LCC-CS, no amplification was observed(Fig. 5A, lane 1). With P2 and LCC-CD, high molecular weightDNA was amplified (Fig. 5A, lane 2). Use of P2 and poly dTresulted in low amplification (lane 3). Interestingly, using P2and LCC-CDT (T: containing a termination codon in the reverseprimer) no amplification was observed (lane 4), nor didamplification result with P2 and LCC-ND (as expected). Withthe specific or degenerate gene sequences of the conserved Nand C termini being used as the primers, no amplification wasobserved. High molecular weight DNA was also amplifiedusing LCC-ND/poly dT primers (lane 7).

About 150 stable E. coli colonies were obtained ontransforming the ligated products of lane 2 and pCR 2.1 vector.Screening with labeled primer LCC-CD resulted in 10 coloniesgiving a positive signal. These were then analyzed forexpression of laccase activity by PAGE–zymogram analysis.The high expression of laccase by one of the clones, pLCC 5, isshown in Fig. 5B, lane 3. The purified laccase of C. bulleri (Fig.5B, lane 1) served as a control and migrated along with theprotein mol wt marker of 66 . The host cells transformed withonly pCR 2.1 vector did not show any activity in the zymogramtest. The laccase expressed in E. coli appeared to be of highermolecular weight compared to the native laccase.

3.6. Partial sequence of the laccase gene

The partial sequence of the laccase gene was determinedfrom a PCR amplified fragment from the reverse transcribed(RT) mRNA. Using the primers P3 and P4 (see Materials andmethods), a DNA fragment of approximately 435 bp wasamplified from the RT mRNA and from the reaction product oflane 2, Fig. 5A. Comparison of the nucleotide sequence with thenucleotide sequences in gene databases was made using theBLASTN program [48] which confirmed that the amplifiedsequences were laccase specific. The alignment of the deducedamino acid sequence in the 435 bp fragment with thecorresponding laccase sequences (based on highest aminoacid sequence similarity result from NCBI search) from other

Fig. 6. Alignment of the deduced amino acid sequence of the PCR amplified 435 bp DNA fragment with the laccase sequences from Coprinopsis cinerea, Coriolopsisgallica, Funalia trogii, Pholiota nameko, Pleurotus pulmonarius, Pleurotus sajor-caju, Phlebia tremellosa, Rigidoporus microporus, Schizophyllum commune.Alignment was done with Clustal V program [46]. The program introduces gaps wherever necessary to maximize matches. Matching amino acids are shown in greycolor.

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fungi is shown in Fig. 6. Maximum similarity (71%) was seenwith Laccase 3 from Coprinopsis cinerea [49] and laccase fromRigidoporus microporus [50] followed by other fungi (60–68%) which included Coriolopsis gallica [51], Funalia trogii[52], Pholiota nameko [53], Pleurotus pulmonarius [54], Pleu-rotus sajor-caju [55], Phelebia tremellosa [56], Schizophyllumcommune [57]. The sequences included the conserved copper-binding domain II of laccases.

4. Discussion

The aim of the present study was to structurally andcatalytically characterize the purified laccase from C. bulleri.This fungus was chosen as it is ecologically specialized in thebreakdown of plant components and has been reported to be aselective lignin degrader. It produces sufficiently high amountof laccase (90 U/ml) which has been shown to be very effectivein decolorization of a large number of reactive dyes [32]. Therange of dyes decolorized was extended in the presence of the

redox mediator ABTS. The products of degradation of severaldyes were also found to be non-toxic for bacterial growth [34].This prompted us to further investigate this enzyme.

On the basis of similarity of three internal peptides of thepurified laccase with other fungal laccases, it was concluded toshare significant similarity with these but on the basis ofexhibiting two different pH optima towards ABTS andguaiacol, the enzyme appeared to be somewhat different. Theamino acid composition data indicated the mol% of chargedamino acids and their amides (Asn, Gln) to be similar to thosepresent in generic laccases but the proportion of hydrophobicamino acids was found to be lower in the C. bulleri laccase. Thecontent of the secondary structure was estimated whichindicated that it had higher proportion of helical content andthe CD fit the α+β structure observed with synthetic peptidescontaining 37% α-helix and 26% β-sheet. This was differentfrom the solved structures of several laccases. These enzymes(with the full complement of copper atoms) consist mainly ofantiparallel β-barrels as observed in the T. versicolor [58–59],

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Melanocarpus albomyces [60], Rigidoporus lignosus [61]enzymes. This was also found in the copper depleted laccasestructure of Coprinus cinereus [62]. In the T. versicolor enzyme[59], the third domain has the highest helical content with one310-helix and two α-helices located in the connecting regionbetween the strands of the different β-sheets. This region in thedomain forms the cavity in which the type-1 copper is located.However, the overall percentage of amino acids in the helicalconformation was much lower for the T. versicolor enzymecompared to the laccase of C. bulleri. From our observations onthe CD spectra, we expect this laccase to belong to α+βcategory and be structurally different from other laccases.

The purified laccase was less sensitive to metal chelation byEDTA. In general, the inhibition by various chemical inhibitorswas similar to that exhibited by the P. cinnabarinus enzyme[36]. One of the important findings in our work is the definitiveinvolvement of a carboxylic group [Asp/Glu] in laccase activitybased on modification with EDC. The drop in enzyme activityat pH values lower than 3.0 was attributed to enzyme instabilitywhile the drop in activity at pHN4.5, was due to ionization of acritical Asp/Glu residue, as indicated by the pKa2 value. Thegroup may have a critical structural or catalytic role asconfirmed by the results of Fig. 4A, where a strongly reactingcarboxylic group was observed leading to nearly 50% loss ofenzyme activity. The order of the reaction showed that onemolecule of EDC bound to one molecule of the enzyme, wheninactivation occurred. Based on sequence comparison of severallaccases of Ascomycete and Basidiomycetes, we have alsolocated an invariant Asp which could have an importantcatalytic role (to be published).

Our results with unfolding and folding of C. bulleri laccasegave some interesting insights into this metallo-enzyme forwhich no information is available. The purified laccase wasdenatured using EDTA at a concentration of 1 mM and with acombination of the denaturants, complete loss of activity wasobserved. The refolding of the protein was done with theaddition of copper under alkaline conditions. The whole processwas completed in 10 min and activity was regained to 100%.The details of the experiment also indicated that addition ofcopper in the initial stages of folding hindered the foldingprocess. Apparently, formation of an apo-enzyme (otherwisecomplete except for the metal ion) was necessary which wasfollowed by final folding and activity regain on addition ofcopper. The absolute requirement of copper was not found to benecessary as zinc (which has a similar atomic radius to copper)was equally effective in restoring enzyme activity. Thus, aflexible role of the metal ion is indicated in laccase activity.

In this paper, we also describe a modification of the LA-PCRapproach for successful expression of laccase. The size of theexpressed laccase was larger than the purified laccase, due toadditional amino acids which may have been added due toexclusion of a termination codon in the designed primer. Theexact size of the laccase expressed in E. coli could not bedetermined as the protein samples were loaded without boilingthe sample. Either extra stability may have been conferred onthe laccase due to addition of extra amino acids or removal of apart of the C-terminus may lend stability to the product. It has

been shown recently that when the C-terminus of the laccase ofM. albomyces was removed from the expression construct,laccase production was considerably improved (six-fold)compared to the full length construct [29]. The expression ofC. bulleri laccase in E. coli appeared to be under control ofthe lac promoter. The partial sequence from the C-terminus ofthe laccase was obtained which indicated that this part had fairlyhigh (60–71%) sequence similarity with other laccases.However, the laccase sequence was sufficiently (about 30%)different also, as being indicated from the amino acidcomposition data.

In conclusion, some interesting and novel structural proper-ties of a laccase are described in this paper which indicate the“not so” critical role of copper atoms and important role ofcarboxylic group containing amino acids. This study alsodescribes novel extension of the LA-PCR approach for generalexpression of enzymes. It may be possible to produce otherlaccases in E. coli as well facilitating the study of these enzymesand their improvement for wider practical applications.

Acknowledgement

This work was supported by a grant from Department ofBiotechnology, Govt. of India, to one of the authors (S.M.).

Appendix A

The SWISS PROT ID of enzymes used in the study fordetermining amino acid composition of laccases from white rotfungi were: Q9UVQ2 (LAC I), Q9HDS9 (LCC3-1), O60199(XA1B), Q99044 (LCC1_TRAVI), Q02497 (LAC 1-TRAHI),Q8TFL8 (072-1), Q8TFM1 (Laccase III), Q8TG94 (Laccase2), Q96UT7 (LAC1), O94222 (LCC2), O13448 (CVL3),Q9P8G4 (LCC1), Q9HDQ0 (LCC1), Q8J1Y2 (LCC1),Q96TR6 (LCC1), Q96VA5 (LCC1), O61263 (Bilirubinoxidase), Q99056 (LCC5), Q12717 (LAC5-TRAVE),O13456 (CVLG1), Q8TG93 (LAP1A), Q9UVQ5 (LAC1),Q9Y781 (LCC2), Q12571 (Laccase), O13421 (POX2),O13422 (POX3), Q8WZH9 (LELCC2), O74171 (Laccase),Q99046 (LCC2),Q12718 (LCC2),Q96UK8 (LAC1),Q9Y782(LCC3), Q9Y780 (LCC1), Q9HDS7 (LCC3-3), Q12739(POX2), Q12729 (POX1), Q9UVY4 (LCCK), Q8WZI0(LELCC2), Q8X1W3 (LCC2), Q9HFT4 (LAC4), Q12719(Laccase 4), CAD 45377.1 (Laccase 1), AAF 06967.1, AAL89554.1 (Laccase), Q99055 (Laccase).

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