GS

MAa

b

Dc

d

a

ARRAA

KLY4GID

1

ifsm[sr[tcso

Dv

h0

Enzyme and Microbial Technology 58–59 (2014) 1–7

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

j o ur na l ho mepage: www.elsev ier .com/ locate /emt

lycosylated yellow laccases of the basidiomycetetropharia aeruginosa

aurycy Darocha,d, Catharine A. Houghtona, Jonathan K. Moorea, Mark C. Wilkinsona,ndrew J. Carnell c, Andrew D. Batesa, Lesley A. Iwanejkob,∗

Institute of Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, United KingdomInstitute of Ageing and Chronic Disease, Faculty of Health & Life Sciences, Department of Musculoskeletal Biology, University of Liverpool,uncan Building, Daulby Street, Liverpool L69 3GA, United KingdomDepartment of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD, United KingdomSchool of Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen 518055, China

r t i c l e i n f o

rticle history:eceived 10 October 2013eceived in revised form 29 January 2014ccepted 5 February 2014vailable online 14 February 2014

eywords:

a b s t r a c t

Here we describe the identification, purification and characterisation of glycosylated yellow laccase pro-teins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellowlaccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins ofmolecular weight around 55 kDa with substrate specificities typical of laccases, but lacking the absorptionband at 612 nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptomesequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA

accaseellow laccase54 pyrosequencingene isolation

nverse PCRye decolorisation

sequences has been assigned to the Yel1p protein on the basis of identity between the translated proteinsequence and the peptide data from the purified protein, and the full length gene sequence has beenobtained. Biochemical properties, substrate specificities and protein sequence data have been used todiscuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theoriesabout the differences between yellow and blue laccases.

© 2014 Elsevier Inc. All rights reserved.

. Introduction

Oxidoreductases (E.C.1) comprise a superfamily of enzymesncluding fungal lignin degrading enzymes. Oxidoreductases arerequently used in industry to catalyse a wide array of reactionsuch as asymmetric oxyfunctionalisations, polymer synthesis andodification, oxidative degradation of pollutants and many others

1]. However, most of these reactions require expensive co-factorsuch as NAD, NADP and FAD, which must be either supplied oregenerated, adding additional steps and further increasing costs2,3]. Laccases (E.C. 1.10.3.2) are oxidative enzymes belonging tohe family of multicopper oxidases that do not require expensive

o-factors; instead they catalyse a one electron oxidation of aubstrate coupled with a four electron reduction of molecularxygen to water. The enzymes use molecular oxygen, rather than

Abbreviations: ABTS 2,2′ , azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); l-OPA, L-3,4-dihydroxyphenylalanine; ConA, Concanavalin A; CBS, Centraalbureauoor.∗ Corresponding author. Tel.: +44 1517064663.

E-mail addresses: iwanejko@liverpool.ac.uk, iwanejko@liv.ac.uk (L.A. Iwanejko).

ttp://dx.doi.org/10.1016/j.enzmictec.2014.02.003141-0229/© 2014 Elsevier Inc. All rights reserved.

peroxide, as an oxidant, and they thus have the capacity to be moreenvironmentally-friendly. This potential for reduced costs alongwith the green credentials of laccases and their ability to utilise arange of substrates, including phenolics, lignins and alkenes, makethem attractive alternatives to other oxidoreductases in a range ofprocesses such as bioremediation and synthetic chemistry.

Laccases are one of the oldest-known group of enzymes andwere discovered as a component of the resin ducts of the lacquertree Rhus venicifera by Yoshida at the end of 19th century [4]. It isnow known that laccases are a diverse group of proteins that can befound in fungi, plants, bacteria and insects [5,6]. Most laccases areblue, resulting from a charge transition from the sulphur atom of acysteine residue to a Cu2+ ion at the catalytic T1 site giving rise to anabsorption band at around 600 nm [7]. However, it is now knownthat not all laccases are blue and both yellow and white laccaseshave also been identified, along with other unusual laccases suchas heterodimeric enzymes [8]. Unlike the four copper atoms foundin blue and yellow laccases, white laccases contain one copper, two

zinc ions and one iron ion per protein molecule [9]. Yellow laccasesare believed to have greater industrial potential than blue laccasesbecause of expanded substrate specificity, often giving them thecapacity to oxidise non-phenolic lignins [10]. There are several

2 M. Daroch et al. / Enzyme and Microbial Technology 58–59 (2014) 1–7

Table 1Protein purification steps used to isolate the Yel1p and Yel3p S.aeruginosa yellow laccases.

No. Purification step Laccase annotation Laccase specific activity (U mg−1) Enrichment

1. Extract 0.60 –2. Salting out 9.50 163. Q Sepharose 16.6 284. Concanavalin A 156 2605. Phenyl Toyopearl flow-through 0.8 M (NH4)2SO4 Yel1p 158 2636. Phenyl Toyopearl elution 0.28 M (NH4)2SO4 198 3307. Phenyl Toyopearl elution 0.064 M (NH4)2SO4 Yel3p 120 200

p

p

p

tEicreccp[m

lTewpicoo

2

2

(2(2

2

sbfsawtABaCi(cT(0cct(wf0

Partial gene sequences of yellow laccases were identified using transcriptomesequencing. Inverse-PCR was then used to obtain the full length cDNA sequencesusing primers based on the partial DNA sequence. The full length cDNA was thenassembled, amplified and cloned into pJET 1.2 vector and sequenced. Unless stated

Table 2Decolourisation of dyes by purified S. aeruginosa yellow laccases Yel1p and Yel3p.

Dye � (nm) Decolourisationby Yel1p after6 h (%)

Decolourisationby Yel3p after 6 h(%)

Amaranth 522 5 8New coccine 506 7 13Orange G 475 8 31

8. Mono Q 0.4 M NaCl Yel19. Superdex 75 Yel1

10. Superdex 75 Yel3

heories attempting to explain the yellow colour of certain laccases.arly reports proposed that yellow laccase proteins are essentiallydentical to blue laccases, but with a lignin-derived mediator in theatalytic site that causes the disappearance of the blue colour and isesponsible for the expansion of substrate specificity [10,11]. How-ver, most recent reports suggest that alterations to the catalyticopper coordinating sphere are the likely explanation for the yellowolour [12]. In addition to laccases, yellow variants of other copperroteins, namely multicopper oxidases have also been described13]. These results show that yellow forms of copper proteins are

ore common than first thought [13].We had previously identified a potentially interesting laccase-

ike activity in the basidiomycete fungus Stropharia aeruginosa.hus our first objective was to purify and characterise thenzyme(s) responsible for this activity [14]. The second objectiveas to clone and characterise the gene coding sequence(s). As in arevious study [15], we employed 454 transcriptome pyrosequenc-

ng to identify the cDNA sequences when more usual PCR-basedloning methods proved unsuccessful, probably as a consequencef gene duplications, the multiplicity of small exons and frequencyf alternative transcripts.

. Materials and methods

.1. Organism

S. aeruginosa CBS 839.87 purchased from Centraalbureau voor SchimmelculturesNetherlands) was cultured in high nitrogen content medium (HNC) containing:% (w/v) glucose, 0.5% (w/v) mycological peptone (Oxoid), 0.2% (w/v) yeast extractSigma), 0.1% (w/v) KH2PO4, 0.05% (w/v) MgSO4, 6 ppm (w/v) NaCl, stationary, at5 ◦C, for four weeks.

.2. Protein purification and biochemical characterisation

Protein was isolated directly from the culture medium, protein purificationteps are summarised in Table 1. The medium was filtered through Miracloth (Cal-iochem) and protein was precipitated overnight with 35% (w/v) (NH4)2SO4 at 4 ◦Crom 5 L of HNC medium. Precipitated protein was pelleted by centrifugation, dis-olved in 100 mM potassium phosphate, pH 6.5 (buffer A) and dialysed overnightgainst buffer A. The solution was then mixed on a roller mixer as a 1/5 (v/v) mixtureith pre-equilibrated Q Sepharose (Sigma) for 30 min to uniformly bind pigment to

he anion exchanger. Unbound protein was recovered by centrifugation and stored.fter washing, bound protein was eluted from the Q Sepharose using 0.5 M NaCl inuffer A, recovered by centrifugation from the pigmented chromatographic mediumnd combined with the unbound fraction. The resultant protein was loaded ontoonA Sepharose (Sigma) equilibrated with 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2

n buffer A. Bound proteins were eluted with 100 mM �-d-methylmannopyranosideSigma) in buffer A. The ConA eluate was saturated with (NH4)2SO4 to a final con-entration of 0.8 M. Precipitated proteins were pelleted at 20,000 × g and discarded.he supernatant was loaded onto Phenyl Toyopearl (Tosoh), equilibrated with 0.8 MNH4)2SO4 in buffer A. Bound proteins were eluted with a decreasing gradient.8 M–0 M (NH4)2SO4. Active fractions were pooled, concentrated with spin con-entrators (Millipore) and denoted as Yel3p. The non-bound fractions (Yel1p) wereollected, pooled, concentrated with spin concentrators and the buffer exchanged

o 100 mM Tris–HCl pH 9.0 (Buffer B). Yel1p was loaded onto a pre-packed MonoQGE Healthcare) chromatographic column equilibrated with buffer B. The proteinas eluted with a gradient of 0–2 M NaCl in buffer B. Protein polishing was per-

ormed with Superdex 75 (GE Healthcare) equilibrated with buffer A containing.15 M NaCl. Molecular weights were determined with the same column calibrated

323 539394 657191 318

with gel filtration molecular weight markers 12,000–200,000 (Sigma). All purifica-tion steps from the ConA step onwards were performed using a BioLogic DuoFlowsystem (Bio-Rad). Protein assays were performed according to the Bradford method[16] using a calibration curve prepared for bovine serum albumin (BSA). SDS-PAGEwas performed according to the method of Laemmli [17].

Purified protein samples (5 �g) were digested with 200 ng trypsin (Sigma) in50 mM ammonium bicarbonate containing 2 M urea overnight at 30 ◦C and stoppedwith 1% (w/v) formic acid. The samples were run on a tandem mass spectrometer atthe University of Liverpool proteomics facility; the data were sequenced manuallyusing the PepSeq module of the MassLynx package (Waters).

2.3. Biochemical analysis

2.3.1. Laccase activity, kinetic analysis, absorbance spectrumOne unit of laccase activity was defined as the amount of enzyme required to

oxidise 1 �mol of substrate per minute. Enzyme activity was determined in trip-licates via the monitoring of the oxidation of a variety of substrates at 42 ◦C for15 min. The assay mixture (1 mL) under standard conditions contained: 890 �L of0.1 M McIlvaine buffer pH 3.0; 100 �L of substrate at 1 mM final concentration and10 �L of appropriately diluted enzyme. The following molar extinction coefficientswere used to determine activity with the various substrates: ABTS 36,000 M−1 cm−1;o-dianisidine 11,300 M−1 cm−1; and syringaldazine 65,000 M−1 cm−1. All mea-surements were made using a Unicam UV2 UV/vis Spectrometer. The kineticmeasurements included variable concentrations of the substrates. Thermostabilitytests included a pre-incubation step of 1 h at temperatures in the range 4–90 ◦C,whereas pH stability included pre-incubation step of 24 h in 0.1 M Britton Robinsonbuffer in the pH range 2–12 at 4 ◦C before the activity measurements were per-formed under standard conditions. The assays used to determine the temperatureactivity profiles were performed over the temperature range 4–80 ◦C. pH activityprofiles were performed in 0.1 M Britton–Robinson buffer in the pH range 2–12.Other parameters of the activity assay were kept to standard conditions. Kinetic datawere calculated on the basis of non-linear fitting with Graphpad Prism 5 (Graph-pad software). The absorbance spectra of purified proteins were measured with aNanodrop 1000 (Thermo Scientific) using 1 �L of purified protein sample.

2.3.2. Dye decolourisationDecolourisation was determined in triplicate by spectrophotometric monitoring

of the oxidation of different dyes (Sigma). Dyes and the wavelengths correspond-ing to their maximal absorbance are summarised in Table 2. Each decolourisationreaction contained 1 mL of 50 �M dye in 0.1 M Britton–Robinson buffer pH 3.0 and0.1 UABTS of purified protein. Decolourisation reactions were performed at 42 ◦C anddecrease in absorbance was monitored at hourly intervals over a 6 h period.

2.4. Molecular biology methods

Tartrazine 428 8 11Remazol Brilliant Blue R 592 19 59Reactive Black 5 590 12 19Reactive Orange 16 494 7 13

icrobial Technology 58–59 (2014) 1–7 3

ot

tdDsruC

owfiais

msftDemsT–aeilD4wN

spsamPAC1

3

3

amsstaptlm

YTtapdmpmrp

Fig. 1. Molecular weight determination of purified S. aeruginosa laccases using SDS-

M. Daroch et al. / Enzyme and M

therwise, all steps using commercial kits were performed according to manufac-urers’ instructions.

Transcriptome sequencing – S. aeruginosa was grown as for the protein isola-ion and the mycelia harvested. Total RNA was isolated according to the methodescribed by Chomczynski [18] and mRNA was purified using Dynabeads® mRNAIRECTTM Kit (Invitrogen). For transcriptome sequencing a SMARTTM cDNA Synthe-

is Kit (Clontech) was used with 1 �g of pure mRNA isolated from cultures. Theesultant library was applied to a matrix for pyrosequencing, which was carried outsing a 454 GS-FLX Titanium pyrosequencer and 1/16 of a sequencing plate at theentre for Genomic Research of Liverpool University.

Sequencing data analysis – after quality control of the pyrosequencing data, a totalf 19,991,305 bp of sequence data was obtained and assembled into 4420 contigsith an average length of 463 bp. BLASTX was used to analyse the contigs in FASTAle using a PC according to the guidelines drawn on NCBI website. This generated

text file which was then searched using search terms “laccase” and “oxidase” todentify contigs that had been identified by the BLASTX search as showing sequenceimilarity to known oxidoreductases.

Inverse PCR – A cDNA library was constructed from the pure sample of theRNA (5 �g) using a ZAP-cDNA Synthesis Kit (Stratagene). The phage library was

ubsequently converted into a plasmid library in SOLR cells. Inverse PCR was per-ormed on the plasmid cDNA library essentially as described by Ochman [19] using aouchdown approach [20]. The reaction was performed in a heated lid thermocyclerNA Engine DYAD Peltier (Bio-Rad) using Phusion pfu polymerase (NEB) and PCRnhancer (Stratagene). The inverse-PCR primers are shown in Supplementary Infor-ation 1 and were derived from all contig sequences identified from the BLASTX

earch as showing sequence similarity to laccases (Supplementary Information 1).he cycling conditions were 98 ◦C for 30 s followed by 5 cycles of 98 ◦C – 10 s; 62 ◦C

30 s; 72 ◦C – 3 min 30 s, followed by identical steps except the annealing temper-ture was decreased each step by 1 ◦C until a Ta of 57 ◦C was achieved. The finalxtension step was performed at 72 ◦C for 10 min; samples were stored at 4 ◦C. Thenverse-PCR product was separated on a 1% (w/v) agarose gel and discrete bandsarger than 4 kbp were excised with a QIAEX II gel extraction kit (Qiagen). IsolatedNA fragments were re-circularised by ligation with T4 DNA ligase overnight at◦C and transformed into E. coli DH5� cells (Bioline). Plasmids were then isolatedith QIAprep Spin Miniprep Kit (Qiagen) and sequenced (Genomes Enterprise Ltd,orwich Research Park).

Laccase cloning – After sequencing the inverse-PCR plasmids the resultantequences were combined with contig sequences and singleton reads from 454yrosequencer with BioEdit to assemble full length sequences of laccases. Laccaseequences were then amplified from genomic DNA isolated according to Raedernd Broda [21], and plasmid cDNA and were cloned into a pJET 1.2 vector (Fer-entas). The PCRs were performed in a heated lid thermocycler DNA Engine DYAD

eltier (Bio-Rad) using Phusion Pfu polymerase (NEB) using the primer pair Yel1 FwdTGCAAGTGCAAGCTCTACTCCGATTC and Yel1 Rev TTAGTCCCTGCAGAGATTCTCC-AAGC. The cycling conditions were 98 ◦C for 30 s and then 30 cycles of 98 ◦C –0 s; 60 ◦C – 30 s; 72 ◦C – 1 min; final extension 72 ◦C – 10 min and hold in 4 ◦C.

. Results and discussion

.1.1. Purification of two laccases – Yel1p and Yel3p

Initial investigations of S. aeruginosa found multiple laccasectivities in the culture medium when grown on HNC medium,easured using the oxidation of ABTS [14], a typical synthetic sub-

trate used for laccase activity assays [5,6,22] and o-dianisidinetaining of native PAGE gels [14]. From multiple laccase pro-eins, two secreted, monomeric, N-glycosylated proteins Yel1pnd Yel3p, of molecular mass approximately 55 kDa were isolated,urified using chromatographic methods and characterised. Fur-her information about other partially characterised S. aeruginosaaccase proteins and genes are described in Supplementary Infor-

ation 2.A number of steps were employed to purify the two laccases,

el1p and Yel3p, from the culture medium, as summarised inable 1. The proteins were isolated from S. aeruginosa HNC cul-ure media after 28 days, the optimal growth period for enzymectivity (0.6 UABTS per mg of secreted protein). During the 28 dayeriod the culture also underwent a colour change from beige toark brown, due to the secretion of a dark pigment most likely ofelanin origin [23] (data not shown). Initial ammonium sulphate

recipitation resulted in the co-precipitation of a dark brown pig-ent, inhibiting isolation of the protein. Precipitated protein was

esuspended, dialysed and mixed in batch with Q Sepharose forigment adsorption and removal. Proteins bound to Q Sepharose

PAGE. Yel1p and Yel1p laccases, after the final protein purification steps, are shownalongside molecular weight markers. 10 �L of each sample was run on a 12% poly-acrylamide gel. The gel was subsequently stained with gel code blue protein stain.

were eluted with 0.5 M NaCl in Buffer A and pooled with unboundfractions, whilst the pigment remained bound to the Q Sepharose.Combined fractions were affinity purified using ConA affinity chro-matography to yield almost ten-fold enrichment of activity. ConAeluate was saturated with ammonium sulphate and loaded ontoa hydrophobic interaction column (Phenyl Toyopearl). The PhenylToyopearl unbound fraction was then buffer exchanged and furtherpurified on a MonoQ column, followed by size exclusion chro-matography on Superdex 75 with the resultant purified proteinbeing designated as Yel1p. The Phenyl Toyopearl bound proteinswere eluted with a gradient of (NH4)2SO4 before polishing onSuperdex 75; the resulting protein was designated Yel3p. Molecu-lar weight determination of both Yel1p and Yel3p was performedusing gel electrophoresis (Fig. 1) and Superdex 75 calibrated withmolecular mass markers. This combination of methods has revealedthat both proteins are monomers of molecular mass of approxi-mately 55 kDa. Purified proteins were subjected to tryptic digestionand peptide sequencing. Yel1p protein contained a unique proteinsequence GPSTNLFIENK, whilst no reliable peptide sequencing datawas obtained for Yel3p.

3.1.2. Biochemical properties of Yel1p and Yel3p

Biochemical analysis of purified Yel1p and Yel3p (summarisedin Fig. 2 and Table 3) confirmed that they both had characteris-tics typical of laccases [5,6]. The temperature and pH optima andstabilities of Yel1p and Yel3p were ascertained using ABTS as thesubstrate. Both proteins exhibited maximum activity at 40 ◦C and

were thermostable up to 40 ◦C (Fig. 2A and B), which are com-mon values for mesophilic laccases [24]. The pH optimum of pH3.0 and acidic range of activity (Fig. 2D) are also characteristic offungal laccases. The pH stabilities of both proteins are also similar

4 M. Daroch et al. / Enzyme and Microbial Technology 58–59 (2014) 1–7

F ty (A)( ed usp ments

tsi(

toupnotasaotsdrtYYcoarr

TSi

ig. 2. Temperature and pH optima of Yel1p and Yel3p laccases. Thermostabiliblack rhombes) and Yel3p (white circles) enzymes. All experiments were performH/temperature. Each data point represents the mean of three independent experi

o their counterparts in other organisms [6], which are generallytable at a pH 5.5 to 9.0, although Yel3p had a narrower pH stabil-ty range (approximately pH 5.0–9.0) than that observed for Yel1papproximately pH 4.0–12; Fig. 2C).

Yel1p and Yel3p showed typical laccase specificities for ABTS,etramethylbenzidine and o-dianisidine. The highest activity wasbserved with the synthetic substrate ABTS which is typicallysed to screen for laccase activity [5,6,12,22,24,25]. These com-ounds can also be substrates for tyrosinases but neither Yel1por Yel3p demonstrated any tyrosinase activity against L-DOPAr L-tyrosine. Both proteins demonstrated specific inhibition withhe low molecular weight compounds NaN3, thiols, ascorbic acidnd L-cysteine typical of laccases [6]. To further test the substratepecificity of the purified laccases, enzymatic decolourisation of azond anthraquinone dyes was performed. The two purified laccasesf S. aeruginosa showed slightly different substrate specificity forhese dyes (Table 2). Both enzymes were most active in decolouri-ation of anthraquinone dye Remazol Brilliant Blue R, the degree ofecolourisation of this dye was 19% and 59% for Yel1p and Yel3pespectively after six hours of decolourisation. Decolourisation ofhe recalcitrant azo dye Reactive Black 5 showed a similar trend,el1p laccase decolourised 12% of the dye in six hours whereasel3p protein 19%. Reactive Black 5 is a high redox potential dyeommonly used for the determination of the industrial potential

f laccases [12,26]. These results show that although laccases of S.eruginosa (especially Yel3p) are capable of decolourising the highedox dye in the absence of exogenous mediators, the rates of theseeactions are low and tend to drop as the reaction proceeds, most

able 3ummary of kinetic data for the purified S. aeruginosa yellow laccases Yel1p and Yel3p. Andependent experiments.

Substrate pH Vmax (�mol min−1)

Yel1po-DA 2.5 117

ABTS 3.0 285

Syringaldazine 4.0 25.4

Yel3po-DA 2.5 1670

ABTS 3.0 255

Syringaldazine 4.0 18.5

temperature optimum (B); pH stability (C) and pH optimum (D) of the Yel1ping ABTS as a substrate. The activity value of 100% represents activity at optimal.

likely due to enzyme inactivation in acidic pH. Overall, the laccasesof S. aeruginosa show typical substrate specificity and do not exhibitexpanded substrate specificity with respect to commonly reportedlaccase enzymes, at least among the substrates and dyes tested.

3.1.3. Cloning and analysis of Yel1 laccase gene sequence

Classical approaches to identifying cDNAs for any of the S.aeruginosa laccase proteins were unsuccessful and it was thereforedecided to sequence the transcriptome using a DNA pyrosequencer.Messenger RNA was isolated from S. aeruginosa culture mediumgrown under optimum laccase expressing conditions. The mRNAwas used to prepare a ZAP-cDNA library and a SMARTTM cDNAlibrary; the latter was sequenced in the Centre for GenomicResearch, University of Liverpool using a 454 GS-FLX Titaniumpyrosequencer. The sequencing run resulted in 4420 contigs assem-bled from the 19,991,305 bp sequenced. The resultant contigswere searched, using a local BLASTX, for laccase sequences. Eightfragments of putative laccase sequences were identified in theS. aeruginosa transcriptome. These contigs were used to designprimer pairs for inverse PCR that were designed to match onlythe most reliable regions of the contigs; attempts were made toavoid homopolymer regions (Supplementary Information 1) thatare problematic when using 454 technology [27]. The primers

were then used in inverse-PCR reactions with the ZAP-cDNA plas-mid library. The resulting inverse-PCR plasmid products weresequenced. Full length laccase sequences were assembled usingBioEdit software with data from inverse PCR, 454 contigs and

ll values were determined at 42 ◦C and given pH and represent the mean of three

Km (�M) kcat (s−1) kcat/Km

1100 14,400 13.1180 35,000 194720 3100 4.37

4500 7600 1.6962 11,500 186

480 830 1.74

icrobia

saawbhTGDYaaaw(dp

3l

wdaatpadcitccll[laAsc(cove

Fc

M. Daroch et al. / Enzyme and M

ingleton reads. From the assembled data, two full length cDNAnd one presumed partial cDNA sequence were identified (summ-rised in Supplementary Information 2). One of these sequencesas presumed to be the coding sequence for the Yel1p protein

ecause it encoded the unique peptide sequence GPSTNLFIENK thatad been identified during analysis of the purified Yel1p protein.he full length cDNA sequence of the Yel1 gene was deposited inenbank (JQ068812). A comparison of the Yel1 cDNA and genomicNA sequences showed the presence of multiple short exons in theel1 gene; Yel1 is composed of 13 exons and 12 introns. Sequencenalysis of the translation product of Yel1 with SignalIP 3.0 [28]nd NetN-Glyc [29] revealed a signal peptide sequence of 21 aminocids and four potential N-glycosylation sites which correspondsith the observed binding of Yel1p to the ConA affinity column

Supplementary Information 3). Due to the lack of protein sequenceata, most likely as a result of protein glycosylation, it was notossible to match any of the cDNA sequences to protein Yel3p.

.1.4. Comparison of Yel1p and Yel3p with fungal blue and yellowaccases

Most laccases are enzymes with a characteristic blue colour,hich is caused by the coordination between cysteine and an oxi-ised copper at site T1 [30,31]. A UV–vis spectrum of purified Yel1pnd Yel3p was performed, which did not show the characteristicbsorption at 612 nm of blue laccases, suggesting that these pro-eins belong to the yellow laccase class (Fig. 3). Moreover, no blueroteins were observed at any point during the purification of Yel1pnd Yel3p, when analysing active protein absorbance at 612 nm. Toate, several laccases showing typical characteristics of blue lac-ases but lacking the characteristic absorption at 612 nm have beendentified [10,32–36], which has prompted a discussion regardinghe origin of these differences. Early reports by Leontievsky ando-workers have shown that the lack of blue colour in yellow lac-ases is not caused by copper loss or sample contamination withignin decomposition products [10]. Firstly, both yellow and blueaccases isolated from the same organisms have similar activities10]; secondly, direct measurements of copper content in both yel-ow and blue laccases have shown that both forms of laccases have

full set of four coppers per laccase molecule [10]. The ratio of280 to A610 was suggested by Leontievsky et al. as a measure ofample purity and an indicator of yellow laccase [10,11]. Both lac-ases of S. aeruginosa exhibit a ratio of A280 to A610 of at least 80Fig. 3), which suggests that these proteins are indeed yellow lac-

ases. Similar ranges of absorbance ratios have been reported forther yellow laccases [10,12,34]. This ratio is also higher than thealue of 36 reported for the yellow laccase of Ganoderma fornicatumxpressed in Pichia pastoris [33].

ig. 3. UV–vis spectrum of purified laccases Yel1p and Yel3p. UV–vis spectra for the puriharacteristic absorption band at 600 nm responsible for the blue colour of blue laccases.

l Technology 58–59 (2014) 1–7 5

Interestingly, yellow laccases often have broader substratespecificities than those seen in blue laccases [11,32,37]. It hasbeen postulated that this expanded substrate specificity originatesfrom binding of aromatic products of lignin degradation to anoriginally blue laccase. A result of this modification could be reduc-tion of copper ions in the active centre of the enzyme causingthe disappearance of the blue colour [10,11]. Two mechanismshave been suggested: direct copper reduction by a free aromaticproduct of lignin degradation, or enzyme binding with such a prod-uct resulting in amino acid modification, conformational changeof the protein and disappearance of the blue colour. The sec-ond hypothesis seemed more likely as yellow proteins have beenfound to be more sensitive towards CO and other inhibitors thantheir blue counterparts, which could be expected from conforma-tional changes around catalytic sites [35,36]. It was also suggestedthat products of lignin degradation can act as electron transfermediators resembling the laccase-mediator system. The mediatormolecule could achieve higher redox potential and expand the sub-strate specificity of some yellow laccases to non-phenolic lignins[11]. Among the substrates tested, the laccases of S. aeruginosashowed substrate specificities typical of blue laccases. Decolouri-sation of textile suggests that although both purified laccases areable to oxidise a number of substrates including high redox azo dyeReactive Black 5, although the rate of this decolourisation is ratherlow without the presence of exogeneous mediators. Similar resultshave been reported for the yellow variant of PM1 basidiomycetelaccase [12], which could not efficiently decolourise Reactive Black5 in the absence of redox mediators.

More recent work by Huang et al. [33] describes the first het-erologous expression of a yellow laccase from G. fornicatum in themethylotrophic yeast P. pastoris. These results suggest that lignindecomposition products are not required for the existence of yellowlaccases as heterologously expressed protein would presumablynot be able to bind to a lignin-derived mediator in a syntheticmedium [33]. In addition yellow variants of a laccase-like multi-copper oxidase expressed homologously in Aspergillus niger grownin minimal medium support this hypothesis [13].

The most recent work of Mate et al. [12] describes directedevolution of high redox potential laccase from the PM1 basid-iomycete in Saccharomyces cerevisiae. During the evolution processan originally blue laccase was changed into a yellow laccase (OB 1variant). Peptide fingerprinting and MALDI-TOF analysis confirmedthe absence of contaminating protein traces that could mask theabsorbance of T1 copper, whereas redox titrations demonstrated

conservation of the redox potential at the T1 site [12]. Moreover,the engineered laccase was cultivated in conventional YP liquidmedium. That raises questions about the requirement of lignin-likedegradation products for the existence of yellow laccases, but does

fied S. aeruginosa yellow laccases, Yel1p (A) and Yel3p (B), showing the lack of the

6 M. Daroch et al. / Enzyme and Microbia

Fig. 4. Multiple sequence alignment of translated protein sequences of S. aeruginosayellow laccase Yel1, with other well-described blue and yellow laccases of fungalorigin. Multiple sequence alignment of the translated protein sequence of the fungalyellow laccase Yel1p from S. aeruginosa (this study), G. fornicatum 0814 DQ914876[33], engineered yellow variant OB1 of basidiomycete PM1 laccase [12] with fungalblue laccases: T. vesicolor 1GYC [38], L. tigrinus 2QT6 [39], C. cinereus 1HFU [40], T.hirsuta 3FPX [41], L. edodes BAB84354 [42] C. unicolor AEQ35306, P. ostreatus POXA1BCAA06291 [43] and original blue laccase from basidiomycete PM1 [12]. Residuesengineered by Mate et al. [12] are marked with an asterix (*). The name of theorganism is followed by accession number in GenBank or Protein Data Bank. Figureprepared with CLC main workbench.

l Technology 58–59 (2014) 1–7

not disqualify the mediator and/or protein modification hypothe-sis altogether. Unlike yellow laccases described in the literature, theevolved yellow laccase OB1 displayed similar catalytic efficienciesas its blue predecessor and could only oxidise high redox poten-tial dyes (Poly R-478, Reactive Black 5, Azure B) in the presence ofan exogenous mediator [12]. Biophysical analysis of purified OB1protein using circular dichroism suggested that polyproline heliceswere formed during the directed evolution, altering spectral prop-erties. In addition, analysis of molecular models indicated that theA461T and S426N mutations, introduced in the T1 environmentduring evolution, appeared to affect the coordinating sphere of theT1 copper [12].

In our study the S. aeruginosa was grown on a synthetic, lignin-free medium. Thus this study also questions the origin of theputative mediator molecule, without disqualifying the hypothesisaltogether. It seems however that if such a mediator molecule hadexisted it would have to be either a chemical present in very sim-ple growth media or could be easily synthesised by yeast and fungifrom basic medium components.

During the evolution of the blue laccase from PM1 basid-iomycete to its yellow form OB1 seven amino acid changes wereintroduced [12]. These amino acid substitutions were mapped ontomultiply-aligned amino acid sequences of S. aerugionsa Yel1, a yel-low laccase of G. fornicatum and sequences of crystallised bluelaccases of T. vesicolor [38], L. tigrinus [39], C. cinereus [40], T. hirsuta[41] and some well described blue laccases L. edodes BAB84354 [42],C. unicolor AEQ35306, P. ostreatus POXA1B CAA06291 [43] (Fig. 4).

Analysis of these protein sequences suggests that there is noobvious pattern corresponding to yellow or blue laccases and thatthe reported individual amino acid substitutions [12] are commonamong both yellow and blue laccases (Fig. 4, marked with asterisk).It is therefore dubious whether any of these amino acid substitut-ions have a direct effect on switching the laccase colour from blueto yellow. It is however possible that their synergistic effect mayresult in alternative folding of the protein and transformation oflaccases from blue to yellow. The alternative polypeptide foldingand formation of polyproline helices may provide an explanationfor the CD spectroscopy results reported before [12]. In our opin-ion more detailed structural studies, most likely involving X-raycrystallography, could provide both verification of the mediatormolecule hypothesis as well as putative alternative folding patternsof yellow laccases.

4. Conclusion

We have shown that S. aeruginosa produces at least two yel-low laccases, which have been purified and characterised. The geneencoding Yel1p has also been characterised. Yellow laccases ofS. aeruginosa show substrate specificities and biochemical prop-erties typical of other laccases. Comparisons of the S. aeruginosalaccase translated gene sequences with other known yellow andblue laccases have failed to identify any clear amino acid motifsthat could explain the differences between yellow and blue lac-cases. It is therefore possible that yellow variants of laccases maybe subjected to alternative folding compared to their blue counter-parts, causing perturbations at the T1 copper site and a change incolour.

Authors’ contributions

MD co-designed and performed majority of experiments, MW

co-designed and performed the experiments, CAH and JKM per-formed the experiments. LAI and AC and ADB supervised theproject and co-designed the experiments. All authors contributedto manuscript preparation.

icrobia

A

faKst

A

i2

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

M. Daroch et al. / Enzyme and M

cknowledgements

This work has been funded from Marie Curie Host Fellowshipsor Early Stage Training MEST-CT-2005-020526 to L.A. Iwanejkond A.D. Bates. Thanks to Prof. Neil Hall, Dr Margaret Hughes and Drevin Ashelford from the Centre for Genomic Research of Univer-ity of Liverpool for 454 sequencing and processing of S. aeruginosaranscriptome samples.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.enzmictec.014.02.003.

eferences

[1] May SW. Applications of oxidoreductases. Curr Opin Biotechnol1999;10:370–5.

[2] Maurer SC, Schulze H, Schmid RD, Urlacher V. Immobilisation of P450BM-3and an NADP(+) cofactor recycling system: towards a technical application ofheme-containing monooxygenases in fine chemical synthesis. Adv Synth Catal2003;345:802–10.

[3] Arnao MB, Acosta M, Delrio JA, Varon R, Garciacanovas F. A kinetic-study onthe suicide inactivation of peroxidase by hydrogen-peroxide. Biochim BiophysActa 1990;1041:43–7.

[4] Yoshida H. Chemistry of lacquer (Urushi). Part 1. J Chem Soc 1883;43:472–86.[5] Giardina P, Faraco V, Pezzella C, Piscitelli A, Vanhulle S, Sannia G. Laccases: a

never-ending story. Cell Mol Life Sci 2010;67:369–85.[6] Baldrian P. Fungal laccases – occurrence and properties. FEMS Microbiol Rev

2006;30:215–42.[7] Lee S-K, George SD, Antholine WE, Hedman B, Hodgson KO, Solomon EI. Nature

of the intermediate formed in the reduction of O2 to H2O at the trinuclearcopper cluster active site in native laccase. J Am Chem Soc 2002;124:6180–93.

[8] Giardina P, Autore F, Faraco V, Festa G, Palmieri G, Piscitelli A, et al. Struc-tural characterization of heterodimeric laccases from Pleurotus ostreatus. ApplMicrobiol Biotechnol 2007;75:1293–300.

[9] Palmieri G, Giardina P, Bianco C, Scaloni A, Capasso A, Sannia G. A novel whitelaccase from Pleurotus ostreatus. J Biol Chem 1997;272:31301–7.

10] Leontievsky AA, Vares T, Lankinen P, Shergill JK, Pozdnyakova NN, MyasoedovaNM, et al. Blue and yellow laccases of ligninolytic fungi. FEMS Microbiol Lett1997;156:9–14.

11] Leontievsky A, Myasoedova N, Pozdnyakova N, Golovleva L. ‘Yellow’ laccaseof Panus tigrinus oxidizes non-phenolic substrates without electron-transfermediators. FEBS Lett 1997;413:446–8.

12] Mate DM, Garcia-Ruiz E, Camarero S, Shubin VV, Falk M, Shleev S, et al.Switching from blue to yellow: altering the spectral properties of a high redoxpotential laccase by directed evolution. Biocatal Biotransform 2013;31:8–21.

13] Tamayo-Ramos JA, van Berkel WJH, de Graaff LH. Biocatalytic potential oflaccase-like multicopper oxidases from Aspergillus niger. Microb Cell Fact2012;11, http://www.microbialcellfactories.com/about.

14] Moore JK. Pursuit of novel haloperoxidase for assymetric biocatalysis. Liver-pool: University of Liverpool; 2007.

15] Florczak T, Daroch M, Wilkinson MC, Białkowska A, Bates AD, Turkiewicz M,et al. Purification, characterisation and expression in Saccharomyces cerevisiaeof LipG7 an enantioselective, cold-adapted lipase from the Antarctic filamen-tous fungus Geomyces sp. P7 with unusual thermostability characteristics.Enzyme Microb Technol 2013;53:18–24.

16] Bradford MM. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein dye binding. AnalBiochem 1976;72:248–54.

17] Laemmli UK. Cleavage of structural proteins during the assembly of the headof bacteriophage T4. Nature 1970;227:680–5.

18] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acidguanidinium thiocyanate phenol chloroform extraction. Anal Biochem1987;162:156–9.

19] Ochman H, Gerber AS, Hartl DL. Genetic applications of an inverse polymerasechain reaction. Genetics 1988;120:621–3.

[

l Technology 58–59 (2014) 1–7 7

20] Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. ‘Touchdown’ PCR to circum-vent spurious priming during gene amplification. Nucl Acids Res 1991;19:4008.

21] Raeder U, Broda P. Rapid preparation of DNA from filamentous fungi. Lett ApplMicrobiol 1985;1:17–20.

22] Alcalde M, Bulter T, Zumarraga M, Garcia-Arellano H, Mencia M, Plou FJ, et al.Screening mutant libraries of fungal laccases in the presence of organic sol-vents. J Biomol Screen 2005;10:624–31.

23] Butler MJ, Day AW. Fungal melanins: a review. Can J Microbiol1998;44:1115–36.

24] Jordaan J, Leukes WD. Isolation of a thermostable laccase with DMAB and MBTHoxidative coupling activity from a mesophilic white rot fungus. Enzyme MicrobTechnol 2003;33:212–9.

25] Rodriguez E, Ruiz-Duenas FJ, Kooistra R, Ram A, Martinez AT, Martinez MJ.Isolation of two laccase genes from the white-rot fungus Pleurotus eryn-gii and heterologous expression of the pel3 encoded protein. J Biotechnol2008;134:9–19.

26] Pereira L, Coelho AV, Viegas CA, dos Santos MMC, Robalo MP, Martins LO.Enzymatic biotransformation of the azo dye Sudan Orange G with bacterialCotA-laccase. J Biotechnol 2009;139:68–77.

27] Moore MJ, Dhingra A, Soltis PS, Shaw R, Farmerie WG, Folta KM, et al. Rapidand accurate pyrosequencing of angiosperm plastid genomes. BMC Plant Biol2006;6, http://www.biomedcentral.com/bmcplantbiol/about.

28] Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signalpeptides: SignalP 3.0. J Mol Biol 2004;340:783–95.

29] Gupta R, Jung E, Brunak S. Prediction of N-glycosylation sites in human proteins.Pacific Symposium on Biocomputing 2002;7:310–22.

30] Bertrand T, Jolivalt C, Briozzo P, Caminade E, Joly N, Madzak C, et al.Crystal structure of a four-copper laccase complexed with an arylamine:insights into substrate recognition and correlation with kinetics. Biochemistry2002;41:7325–33.

31] Shleev SV, Morozova OV, Nikitina OV, Gorshina ES, Rusinova TV, SerezhenkovVA, et al. Comparison of physico-chemical characteristics of four laccases fromdifferent basidiomycetes. Biochimie 2004;86:693–703.

32] Pozdnyakova NN, Rodakiewicz-Nowak J, Turkovskaya OV. Catalytic proper-ties of yellow laccase from Pleurotus ostreatus D1. J Mol Catal B Enzym2004;30:19–24.

33] Huang WT, Tai R, Hseu RS, Huang CT. Overexpression and characterization ofa thermostable, pH-stable and organic solvent-tolerant Ganoderma fornicatumlaccase in Pichia pastoris. Process Biochem 2011;46:1469–74.

34] Pozdnyakova NN, Turkovskaya OV, Yudina EN, Rodakiewicz-Nowak Y. Yellowlaccase from the fungus Pleurotus ostreatus D1: purification and characteriza-tion. Appl Biochem Microbiol 2006;42:56–61.

35] Leontievsky AA, Pozdnyakova NN, Myasoedova NM. Comparative characteriza-tion of oxidase-1 from Panus tigrinus 8-18 and laccase from Coriolus versicolorVKM F-116. Biochemistry (Moscow) 1996;61:1262–8.

36] Pozdnyakova N, Leontievsky A, Golovleva L. Oxidase of the white rot fungusPanus tigrinus 8/18. FEBS Lett 1994;350:192–4.

37] Sahay R, Yadav RSS, Yadava S, Yadav KDS. A laccase of fomes durissimus MTCC-1173 and its role in the conversion of methylbenzene to benzaldehyde. ApplBiochem Biotechnol 2012;166:563–75.

38] Piontek K, Antorini M, Choinowski T. Crystal structure of a laccase from thefungus Trametes versicolor at 1.90-A resolution containing a full complementof coppers. J Biol Chem 2002;277:37663–9.

39] Ferraroni M, Myasoedova NM, Schmatchenko V, Leontievsky AA, Golovl-eva LA, Scozzafava A, et al. Crystal structure of a blue laccase fromLentinus tigrinus: evidences for intermediates in the molecular oxygenreductive splitting by multicopper oxidases. BMC Struct Biol 2007;7,http://www.biomedcentral.com/bmcstructbiol/about.

40] Ducros V, Brzozowski AM, Wilson KS, Østergaard P, Schneider PA, et al. Struc-ture of the laccase from Coprinus cinereus at 1.68 A resolution: evidence fordifferent ‘type 2 Cu-depleted’ isoforms. Acta Crystallogr D Biol Crystallogr2001;57:333–6.

41] Pegasova TV, Zwart P, Koroleva OV, Stepanova EV, Rebrikov DV, LamzinVS. Crystallization and preliminary X-ray analysis of a four-copper lac-case from Coriolus hirsutus. Acta Crystallogr D Biol Crystallogr 2003;59:1459–61.

42] Nagai M, Sato T, Watanabe H, Saito K, Kawata M, Enei H. Purification and char-acterization of an extracellular laccase from the edible mushroom Lentinula

edodes, and decolorization of chemically different dyes. Appl Microbiol Bio-technol 2002;60:327–35.

43] Giardina P, Palmieri G, Scaloni A, Fontanella B, Faraco V, Cennamo G, et al.Protein and gene structure of a blue laccase from Pleurotus ostreatus. BiochemJ 1999;341:655–63.