Preliminary studies of new strains of Trametes sp. from Argentina for laccase production ability

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Preliminary

studies

of

new

strains

of

Trametes

sp.

from

Argentina

for

laccase

production

ability

María

Isabel

Fonseca

∗

,

Marcos

Raúl

Tejerina,

Silvana

Soledad

Sawostjanik-Afanasiuk,

Ernesto

Martin

Giorgio,

Mónica

Lucrecia

Barchuk,

Pedro

Darío

Zapata,

Laura

Lidia

Villalba

LaboratoriodeBiotecnologíaMolecular,MódulodeBioquímicayFarmacia,FacultaddeCienciasExactasQuímicasyNaturales,UNaM, Posadas,Misiones,Argentina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22April2012

Accepted6June2015

Availableonline2March2016

AssociateEditor:MiguelJuan

Beltran-Garcia

Keywords:

Whiterotfungi

Trametes

Laccase

Manganeseperoxidase

Cu2+_response

a

b

s

t

r

a

c

t

Oxidativeenzymessecretedbywhiterotfungicanbeappliedinseveraltechnological

pro-cesseswithinthepaperindustry,biofuelproductionandbioremediation.Thediscoveryof

nativestrainsfromthebiodiverseMisiones(Argentina)forestcanprovideusefulenzymes

forbiotechnologicalpurposes.Inthiswork,weevaluatedthelaccaseandmanganese

per-oxidasesecretionabilitiesoffournewlydiscoveredstrainsofTrametessp.thatarenative

toMisiones.Inaddition,thecopperresponseandoptimalpHandtemperatureforlaccase

activityinculturesupernatantsweredetermined.

TheselectedstrainsproducedvariableamountsoflaccaseandMnP;whenCu2+_was_added,

bothenzymesweresigniﬁcantlyincreased.Zymogramsshowedthattwoisoenzymeswere

increasedinallstrainsinthepresenceofCu2+_._Strain_B_showed_the_greatest_response_to

Cu2+_addition,_whereas_strain_A_was_more_stable_at_the_optimal_temperature_and_pH._Strain

Ashowedinterestingpotentialforfuturebiotechnologicalapproachesduetothesuperior

thermo-stabilityofitssecretedenzymes.

anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

In recent years, many innovative technologies have been

developed to reduce pollution and generate ecologically

friendly energy. Searching for new promising

microorgan-isms with signiﬁcant enzymatic secretion systems has

providedinnovativebiotechnologicalresources.Theprovince

ofMisioneshasoneofthehighestbiodiversitiesinArgentina,

∗ _{Corresponding}_author.

E-mail:biotecmol2010@gmail.com(M.I.Fonseca).

andtogetherwiththeYungasregion,theseregionsarethe

bio-logicallyrichestnaturalareasofArgentina.However,thestudy

offungi,especiallymicrofungi,hasbeenneglected,andmany

fungiawaitextensivestudies.Thiswonderfulnaturalareais

amycologist’sparadise.1_Many_white_rot_fungi_(WRF)_species

growinforestareasofMisionesandproduceacomplex

sys-temofligninolyticenzymeswithbiotechnologicalpotential.

Ligninperoxidase(Lip),manganeseperoxidase(MnP)and

laccase (Lac) are the most widely distributed ligninolytic

http://dx.doi.org/10.1016/j.bjm.2016.01.002

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enzymes.2–4_The_synthesis_of_these_enzymes_is_regulated_by

variousfactors,andheavymetalssigniﬁcantlyaffectenzyme

production.5–7_Temperature_and_pH_also_greatly_inﬂuence_the

activityandthermostabilityofthesecretedenzymes.8

Principallydue tothe polymorphism ofBasidiomycetes,

diversetaxonomicalcriteriahavebeenusedtoclassifythese

fungi; therefore, the correct name for many taxa used in

variousstudieshaveremainedunclear.9,10_However,_the

phy-logeneticdiversityofstrainsisrelatedtotheenvironmental,

ecologic, geographic and geneticconditions inwhich they

live.11 _Molecular _techniques _are _useful _tools _to _study _the

phylogenyand intraspeciﬁcgeneticvariations, andprovide

essential information for understanding enzyme variation

amongstrains ofthe same fungal species.9,12 _The_internal

transcribedspace (ITS)regionsofribosomalDNAhavealso

beenusedtoanalyzevariousgeneraandspeciesoffungi.13–15

The sequences of these regions are more diverse among

species than within species.16 _The _aim _of _this _work _was

to evaluate the laccase and manganese peroxidase

secre-tion abilities of new strains of Trametes sp. isolated from

Misiones, Argentina. The effect of the addition of Cu2+_,

the thermostability, and the optimal pH and temperature

of laccase activity in the culture supernatants were also

analyzed.

Materials

and

methods

Microorganismsandcultureconditions

WhiterotfungistrainsA,B,C,andEwereprovidedbythe

Cul-tureCollectionoftheFacultyofForestry,NationalUniversityof

Misiones,Argentina;thestrainsweresubjectedtomolecular

phylogeneticclassiﬁcationasdescribedbelow.Stockcultures

weremaintainedonmaltagarat4◦C.

Toprepareliquidinocula,eachfunguswasgrownfor5–7

daysonmaltagarplates (MEA:20gL−1agar,12.7gL−1malt

extract),and36-mm2_agar_plugs_were_then_cut_from_the_agar

andtransferredto100-mLErlenmeyerﬂaskscontaining20mL

ofmedium(ME:12.7gL−1maltextractand5gL−1cornsteep

liquor)andincubatedat29◦Cinsteady-stateconditions.

Biomassandproteindetermination

Biomassgrowthwasdeterminedbymeasuringthemycelium

dry weight, and proteinsin the conditioned mediumwere

measuredusingtheBradfordmethod.

Liquid medium was separated from the supernatant

mycelium by filtering through fiberglass filters (GF/C) in a

Büchnerfunnelandfrozenat−20◦_C_until_use._Biomass_dry

weightwasdeterminedbymeasuringthedifferencebetween

thefiberglassfilter(GF/C)weightbeforefiltrationandafter

ﬁl-trationandsubsequentdryingat80◦Cuntilaconstantweight

wasattained.

Proteinwasdeterminedusingamicro-testbasedonthe

Bradford technique (BioRad) following the manufacturer’s

instructions;bovineserumalbuminwasusedasthestandard.

Secreted protein concentrations are expressed in units of

␮g/mL.

Enzymeassays

Laccase(EC1.10.3.2)activitywasmeasuredat30◦Cusing1mL

of5mM2,6-dimethoxyphenol(DMP)in0.1Msodiumacetate

buffer pH 3.6 and 50␮L of ﬁltered supernatant mycelium.

Absorbancewasmonitoredat469nm(E469=27.5mM−1cm−1)

using aShimadzuUV-3600spectrophotometer. Onelaccase

activityunitwasdeﬁnedastheamountofenzymerequired

tooxidize1␮molofDMPperminat30◦Candisexpressedin

unitsofUmL−1.7

Manganese peroxidase (EC 1.11.1.13) activity was

mea-sured at 30◦C using 2.5mLof 0.1M phenol red in sodium

dimethylsuccinatebufferpH4.5and50␮Lofﬁltered

super-natant mycelium. The reaction was initiated using 20␮L

of 0.2mM H2O2. Absorbance was monitored at 610nm

(E610=22mM−1cm−1)usingaShimadzuUV-3600

spectropho-tometer.OneMnPactivityunitwasdeﬁnedastheamountof

enzymerequiredtooxidize1␮molofphenolredperminat

30◦CandisexpressedinunitsofUmL−1.17

Polyacrylamidegelelectrophoresis

To identify the number of isoenzymes involved in Cu2+

induction, thecrude enzymewas subjectedtonative

poly-acrylamide gel electrophoresis (ND-PAGE) and denaturing

polyacrylamide gelelectrophoresis(SDS-PAGE)in7.5%gels.

AfterresolvingtheproteinsusingND-PAGE,thegelwas

incu-batedin0.1Msodiumacetatebuffercontaining5mMDMP

beforedetectinglaccaseactivity.18_After_incubating_the_gel_for

5min,thedyesolutionwasdiscarded;thegelwas

immedi-atelyscanned usinga scanner(HPDeskjetF300All-in-One

series).

Themolecular weight oftheisoenzymes was evaluated

using 7.5%SDS-PAGEincluding amolecular weightmarker

(Kaleidoscope, BioRad).SDSwasremovedbyincubatingthe

gelin50mMsodiumacetatecontaining0.2%TritonX-100and

thenstainingwith5mMDMPtodetectlaccaseactivity.19

Determinationofthethermostabilityandoptimal temperatureandpHoflaccaseactivityinculture supernatants

Thelaccaseactivityintheculturesupernatantswastested

atpH3.6–5.6in50mMsodium–acetatebufferusingDMPas

substrate.AfterdeterminingtheoptimumpH,laccaseactivity

wasmeasuredintherange10–80◦C.

Thethermostabilitywasevaluatedattheoptimalvaluesof

pHandTusingculturesupernatantsthathadbeenincubated

for7h.

DNAextraction

Fungimyceliawereﬁlteredandwashedwith0.1MTris–HCl

pH 8 and 0.02M EDTA. DNA was extracted inbuffer

solu-tion (100mM Tris–HCl pH 8, 1.5M NaCl, 50mM EDTA pH

8) at 60◦C containing 0.1mgmL−1 proteinase K, 10mM

␤-mercaptoethanoland2%SDS.TheDNAwaspuriﬁedwith

chloroform,isoamylalcohol(24:1)and3Mpotassiumacetate

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ITSampliﬁcationandsequencinganalysis

ITS-1(partialsequence),the5.8SrRNAgene,ITS2,andthe

28SrRNAgene(partialsequence)region,(ITS1-5.8S-ITS2-28S),

were ampliﬁed and sequenced; the sequences were then

depositedinGenBank.

The genes were ampliﬁed using the primer sequences

describedbyWhiteetal.16_PCR_was_carried_out_in_20-_␮L

reac-tionscontaining1×KClbuffer,2.5mMMgCl2,200␮MdNTPs,

10pmol ofeach primer, 0.5U Taq polymeraseand 25ngof

DNA. ThePCR program was as follows: 4min at 94◦C; 35

roundsof40s94◦C,40s50◦C,and40s72◦C;andﬁnal

exten-sionfor10minat72◦C.Allfragmentswerethensubmittedto

asequencingservice(Macrogen,Korea).

All sequences were analyzed using Chromas Lite 2.01,

BLASTn, BioEdit and CLUSTAL W before phylogenetic tree

construction.Phylogeneticanalysiswascarriedoutusingthe

T.N.T.program.20 _Gaps_(indels) _were _treated_as _a _5th _state

becausetheyrepresentedinsertion–deletionevents.The

anal-ysisincluded42sequences,andDaedaleopsistricolor,Hexagonia

nitidaand H.mimeteswereusedasoutgroups.4,21,22 _Because

thedatasetwasreduced,theheuristicsearcheswere

imple-mentedusing1000RAS,savingonetreeperTBR.Toassessthe

supportfortheidentiﬁedgroups, bootstrapand parsimony

jack-kniﬁnganalyseswereperformed.23 Boththe bootstrap

andjack-knifeanalysesincluded1000resampledmatrices.For

eachresampledmatrix,weperformed100RAS+TBRcycles.

Statisticalanalysis

Two-wayANOVAandBonferroni’sposttestwereperformed

usingGraphPadPrism4.00forWindows(GraphPadSoftware,

SanDiego,CA,USA).

Results

Culturalcharacteristicsandphylogeneticstudiesofnew Trametesstrains

Inthiswork,weworkedwithfourstrainsofwhiterotfungi

thatwere characterizedasmembers ofthe Trametesgenus.

ThefourstrainsshowedwhitemyceliadevelopmentonMEA

but exhibited differentculturalcharacteristics: strainsA, C

andEshowedcotton-likecolonies,whereasstrainBshowed

colonieswithsmoothersurfaces(Fig.1).

To conduct phylogenetic studies, we determined the

nucleotidesequenceoftheITS1-5.8S-ITS2-28Sregionofstrain

A (538bp), strain B (548bp), strain C (547bp), and strain

E(475bp);eachfungusexhibited97%sequenceidentitywith

Strain A

Strain B

Strain C

Strain E

Fig.1–CulturalcharacteristicsofTrametessp.strainsonMEA.Fungiweregrownfor7daysonmaltagarplates (20gL−1agar,12.7gL−1maltextract).Lettersindicatestraincodes.

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Fig.2–ITSregionof5.8SrDNAphylogeneticrelationships betweenstrains.Groupsupportwasassessedusing1000 bootstrappingandparsimonyjack-kniﬁngreplicates. Numbersabovebranchescorrespondtojack-knifesupport. Bootstrapsupportsaregiveninparentheses.

Trameteshirsuta,TrametesversicolorandTrametesvillosa.Blast

andFungiBarcode(http://www.fungalbarcoding.org)searches

showed that the fungi were closely related to the genus

Trametesamongtheexaminedbasidiomycetes.Basedonthe

resultsofthe Blastsearch,subsequent phylogenicanalysis

was performedusing mainlyspeciesfrom thegenus

Tram-etes. Sequencesobtainedfromallstrains weredepositedin

GenBank underthe followingaccess numbers:HM222419.2

(strainA);HM622150.1(strainB);HM622151.1(strainC)and

HM622153.1(strainE).

Aphylogenictreewasobtainedfromoverlappingdatasets

of the ITS region(Fig. 2).Informative sites (deﬁnedby the

presenceofanygaps,ambiguitysymbols,andnonconsensus

sequenceateachsite)includedthoseat88/612.After

remov-ing poorly alignedpositions anddivergent regions ofDNA,

the ITSsequences were aligned over 612 characters, 88 of

whichwereparsimoniouslyinformative.Parsimonyanalysis

resultedin80equallyparsimonioustreesthatwere185steps

long.Bothbootstrapandjack-knifeprocessesyieldednearly

thesametopology,andtheirsupportvalueswerevery

simi-lar.Theresultingtreesshowedthatallstrainsstudiedinthis

workformedamonophyleticcladethatwascloselyrelatedto

T.villosa.

Biomass,andproteinandoxidativeenzymesecretion

Thebiomassandsecretedproteinintheliquidmediumwere

signiﬁcantly different among the strains (Fig. 3). Strain A

showedasigniﬁcantincreaseinbiomasswithdaysofculture

(p<0.001).All strainssecretedsimilar quantitiesofprotein,

andthehighestcontentswerefoundoncultureday10.

To verify thelaccaseand MnPsecretion patterns ofthe

analyzedstrains,allfungiwereculturedfor7daysinliquid

mediumcontainingmaltextract.Thestrainssecretedlaccase

todifferingextents(Table1).StrainsAandCshowedthe

high-estamountsoftotallaccase,andstrainCshowedthehighest

0.15

A

B

60 40 20 0 6 8 10 12 0.10 0.05 0.00 6 8 10 Culture day References

Strain A Strain B Strain C Strain E Culture day

Biomass (g)

Secreted proteins

(µg/ml)

12 14 14

Fig.3–Biomass(a)andsecretedproteins(b)ofTrametessp.strains.AllstrainswereculturedonMEmediumfor14daysat 29◦CandpH4.8.Biomassaccumulationandamountofsecretedproteinsweredeterminedforallsub-strainsat7,10and14 culturedays.Allexperimentswerecarriedoutintriplicate.

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3500 3000 2500 2000 1500 1250 Enzimatic activity (U mL − 1)

Strain A

Strain B

Strain C

Strain E

1000 750 500 250 7 7 0 0.2 0.5 0 0.2 0.5 0 0.20.5 10 14 10 Culture day Day Cu++ 7 0 0.2 0.5 0 0.2 0.5 0 0.20.5 10 14 Day Cu++ 14 7 10 Culture day 14 7 7 0 0.2 0.5 0 0.2 0.5 0 0.20.5 10 14 10 Culture day Day Cu++ 7 0 0.2 0.5 0 0.2 0.5 0 0.2 0.5 10 14 Day Cu++ 14 7 10 Culture day 14 0 3500 3000 2500 2000 1500 1250 Enzimatic activity (U mL − 1) 1000 750 500 250 0 3500 3000 2500 2000 1500 1250 Enzimatic activity (U mL − 1) 1000 750 500 250 0 3500 3000 2500 2000 1500 1250 Enzimatic activity (U mL − 1) 1000 750 500 250 0

Fig.4–EffectofCu2+_on_laccase_activity_in_the_studied_Trametes_sp._strains._All_strains_were_cultured_on_ME_for₁₄_days_in_the

presenceorabsence()of0.2mM( )and0.5mMCu2+().TheexperimentswerecarriedoutinMEmedium,andenzyme activitywasmeasuredon7,10,and14daysofculture(uppergraphs).Alldeterminationswerecarriedoutintriplicate,and theresultsareexpressedasUmL−1.LaccaseisoenzymesweredetectedusingND-PAGEanalyseswithDMP(lowergraphs).

Table1–LaccaseandMnPsecretionbyTrametessp. strains. Laccase MnP Enzymaticactivity UmL−1 Enzymaticactivity UmL−1 StrainA 190.74±15.33 60.42±29.32 StrainB 78.76±12.74 37.44±19.74 StrainC 325.00±19.05 Undetectable StrainE 19.65±4.21 Undetectable StrainsweregrownonMEundersteady-stateconditionsat29◦C andpH4.5.

Enzymaticactivitiesweremeasuredonday7ofcultureintriplicate. Dataareshownasmedians±SD.Determinationswerecarriedout intriplicate.

productionoflaccaseperunitofbiomassproduction(Ug−1of mycelia).OnlystrainsAandBshoweddetectablequantities ofMnPontheday7ofculture.

EffectoftheadditionofCu2+_on_laccase_and_manganese

peroxidaseactivity

Previouslypublisheddatashowedthatthe additionofCu2+

increases laccaseactivity in Basidiomycetes.To verify that thisalsooccursinthenewlydiscoveredstrains,laccase activ-itywasmeasuredintheabsenceandpresenceof0.2mMand 0.5mMCu2+_._The_experiments_were_carried_out_in_ME_medium,

andenzymeactivitywasmeasuredondays7,10and14of culture(Fig.4).

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Strain A

500 400 300 200 Enzimatic activity (U mL − 1) 150 100 50 0 7 10 14 Culture day 500 400 300 200 Enzimatic activity (U mL − 1) 150 100 50 0 7 10 14 Culture day 500 400 300 200 Enzimatic activity (U mL − 1) 150 100 50 0 7 10 14 Culture day 500 400 300 200 Enzimatic activity (U mL − 1) 150 100 50 0 7 10 14 Culture day

Strain B

Strain C

Strain E

Fig.5–EffectofCu2+_on_MnP_activity_in_Trametes_sp._strains._All_strains_were_cultured_on_MEA_for₁₄_days_in_the_presence_or

absence()of0.2mM( )and0.5mMCu2+().TheexperimentswerecarriedoutinMEmedium,andenzyme

measurementsweremadeondays7,10and14ofculture(uppergraphs).Alldeterminationswerecarriedoutintriplicate, andtheresultsareexpressedasUmL−1.

Theaddition ofCu2+ _led_to_a_signiﬁcant_increase_in

lac-case secretion in all strains (p<0.05). Maximum secretion

occurred upon the addition of0.2mMCu2+ _to_strains _A, _B

andE.StrainCexhibitedmaximallaccasesecretionuponthe

additionof0.5mMCu2+_._Strain_B_responded_most_strongly_to

Cu2+_induction_on_day₇_of_culture_(3085.5_U_mL−1_;_i.e.,_39.5-fold

greaterthanthesecretionwithoutCu2+_(78.7_U_mL−1_))._Strain

ErespondedmoststronglytoCu2+inductiononday10of

cul-ture(1104.5UmL−1);i.e.,33.2-foldgreaterthanthesecretion

withoutCu2+_(33.2_U_mL−1_);_p_<_0.001)._However,_strains_A_and

Cexhibitedonly4.67-foldinductionbyCu2+_._Strain_A_secreted

891UmL−1oflaccaseinthepresenceofCu2+_and₁₉₁_U_mL−1

initsabsence.StrainCsecreted1519UmL−1oflaccaseinthe

presenceofCu2+_and₃₂₅_U_mL−1_in_its_absence.

Aprevious report described that the number oflaccase

isoenzymesvariesamongBasidiomycetes.7 _To_examine_the

presenceoflaccaseisoenzymesandtheirdifferentialresponse

toCu2+_induction,_ND-PAGE_analyses_with_DMP_were_carried

out(Fig.4).Zymogramanalysisshowedaclearincreaseinthe

densityoftwolaccasebandsforallstrainsinthepresenceof

Cu2+_._The_upper_bands_appeared_for_strains_A,_B_and_E_only_in

thepresenceofCu2+_,_whereas_strain_C_showed_an_upper_band

intheabsenceofCu2+_._Using_SDS-PAGE_with_DMP_detection,

weestablishedthemolecularweightofthedetectedlaccase

bands:70kDaforthelowerbandand140fortheupperband.

WealsodeterminedMnPactivitiesunderbasalconditions

without theadditionofexogenousMn2+ _in_the_presence_of

0.2or0.5mMCu2+_._All_strains_showed_a_clear_increase_in_MnP

secretioninthepresenceofCu2+ _(p_<_0.05)._Strain_B_showed

thehighestresponsetotheadditionofCu2+onday7of

cul-tureday;similaramountsofMnPweresecretedusingboth

Cu2+ _{concentrations}_(357.4_U_mL−1_;_9.5-fold_higher_than_that

intheabsenceofCu2+ _(37.4_U_mL−1_))._Strain_A_showed_high

MnPsecretioninthepresenceof0.2mMofCu2+ _on_day₁₀

ofcultureday(102.7UmL−1;1.7-foldhigherthanthatinthe

absenceofCu2+ _(58.6_U_mL−1_))._Strains_C_and_E_only_secreted

MnP(invariableamounts)inthepresenceofCu2+_(Fig._5).

ThermostabilityandtemperatureandpHoptimafor laccaseactivity

WedeterminedtheoptimalTandpHforlaccaseactivityin

culturesupernatantsinthepresenceandabsenceofCu2+_.

Cul-turesupernatantsofallstrainsexhibitedmaximalactivityat

55◦CinthepresenceofCu2+_._In_the_absence_of_Cu2+_,_strains

AandEexhibitedmaximalactivity at55◦C,strainB

exhib-itedmaximalactivityat50◦C,andstrainCexhibitedmaximal

activityat45◦C(Fig.6).

CulturesupernatantslackingCu2+ _did_not_show_any

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6000 4000 2000 150 Enzymatic activity U L − 1 100 50 0 15 20 25 30 35 40 45 Temperature (°C) 50 55 60 65 70 75 6000 4000 2000 150 Enzymatic activity U L − 1 100 50 0 15 20 25 30 35 40 45 Temperature (°C) 50 55 60 65 70 75 80 6000 4000 2000 150 Enzymatic activity U L − 1 100 50 0 15 20 25 30 35 40 45 Temperature (°C) 50 55 60 65 70 75 6000 4000 2000 150 Enzymatic activity U L − 1 100 50 0 15 20 25 30 35 40 45 Temperature (°C) 50 55 60 65 70

Fig.6–EffectoftemperatureonlaccaseactivityinculturesupernatantsofTrametessp.strains.Allstrainswereculturedon MEfor14daysintheabsence(triangle)orpresenceof0.5mMCu2+₍_)._The_experiments_were_conducted_between₁₀◦_and

80◦C.AlldeterminationswerecarriedoutintriplicateusingDMPassubstrate,andtheresultsareexpressedasUmL−1.

supernatantsincludingCu2+exhibitedmaximallaccase

activ-ityatpH3.6;themaximalactivityinthepresenceofCu2+_was

greaterthanthatintheabsenceofcopper(13-foldforstrainA

(p<0.001),300-foldforstrainB(p<0.001),140-foldforstrainC

(p<0.001),and100-foldforstrainE(p<0.001);Fig.7).

To measure the thermostability of the enzyme, culture

supernatantswereincubated for7hatthe optimalpHand

T.Residuallaccaseactivitywasestimatedbasedonthe

high-estvalueobtained(100%).StrainAhadahalf-lifeof300minin

culturesupernatantsincludingCu2+_and_a_half-life_of₁₂₀_min

insupernatantsincludingCu2+_._Strains_B,_C_and_E_had_a

half-life of 40min inthe presenceand in the absence of Cu2+

(Table2).

Discussion

In this work, weevaluated oxidativeenzymes secreted by

four newlydiscovered Trametesstrainsand focusedon

lac-case activity.These strains showed differences inbiomass

Table2–ResiduallaccaseactivityinculturesupernatantsincubatedatoptimaltemperatureandpH.

Time(min) Residualenzymaticactivity

StrainA StrainB StrainC StrainE

With0.5mM ofCu2+ Without Cu2+ With0.5mM ofCu2+ Without Cu2+ With0.5mM ofCu2+ Without Cu2+ With0.5mM ofCu2+ Without Cu2+ 0 100 100 100 100 100 100 100 100 20 91 113 82 92 63 65 81 92 40 55 97 58 61 52 47 69 32 60 59 96 31 58 43 54 36 7 120 56 51 36 13 31 12 23 8 180 52 49 32 14 30 – 18 – 240 48 36 27 – 17 – 10 – 300 56 32 29 – 13 – 10 – 360 40 31 26 – 13 – 6 – 420 45 – 24 – – – 6 –

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4000 3000 2000 1000 150 100 50 0 Enzymatic activity U L –1 pH pH pH pH 3.63.8 4 4.24.44.64.8 5 5.25.45.6 4000 3000 2000 1000 150 100 50 0 Enzymatic activity U L –1 3.63.8 4 4.24.44.64.8 5 5.25.45.6 4000 3000 2000 1000 150 100 50 0 Enzymatic activity U L –1 5.6 5.4 5.2 5 4.8 4.6 4.4 4.2 4 3.8 3.6 4000 3000 2000 1000 150 100 50 0 Enzymatic activity U L –1 3.63.8 4 4.24.44.64.8 5 5.25.45.6

Fig.7–EffectofpHonlaccaseactivityinculturesupernatantsofTrametessp.strains.AllstrainswereculturedonMEfor14 daysintheabsence(triangles)orpresenceof0.5mMCu2+₍_)._The_experiments_were_conducted_in_the_pH_range_3.6–5.6

using0.1Msodiumacetatebuffer.AlldeterminationswerecarriedintriplicateoutusingDMPassubstrate,andtheresults areexpressedasUmL−1.

production,levelsofsecretedproteinsandlevelsofthemain

oxidativeenzymelaccase.

TheITS1 and ITS2 regions, which are separated bythe

conservedshort5.8SrRNA,mayexhibitnucleotidevariations

becausetheirtranscriptsareexcisedfromtheﬁnalrRNA

frag-ments.Consequently,wedecidedtostudythesesequences.

Sequencesarecommonlyusedtoinferphylogenetic

relation-shipsofcloselyrelatedspeciesandtoassessthevariability

presentwithinapopulation;e.g.,amonggeographically

dis-tantisolates(ecotypes).10 _Although_a_phylogenetic _analysis

oftheITS1-5.8S-ITS2-28S regionsrevealed that the studied

strainsare closelyrelatedtothe T.villosacladebranch, we

wereonlyabletoidentifythestrainsatthegenuslevel.T.

vil-losawasrecentlyisolatedinGuadeloupeandArgentina.4_More

extensiveITStreeswere constructedusingall theavailable

ITS1-5.8S-ITS2sequencesofgoodqualitythatwere520–560nt

inlengthfromTrametesspp.andmembersofcloselyrelated

genera(datanotshown);thesetreesindicatethatthesestrains

might representdivergent isolatesof the speciesT. villosa.

Finally,toclarifythetaxonomicpositionofthestrains,itwill

benecessarytocomplementourmolecularstudieswith

clas-sicalgeneticanalysis; e.g.,basedon matingexperiments.24

TheTrametesspeciesidentiﬁedandstudiedinthisworkare

relatedtothethirdcladeobtainedbyWeltietal.,4

compris-ingagroupofspecimensfromTrametespubescenstoT.hirsute.

Three distinct sub-clades have been identiﬁed within this

clade,andthesub-cladeofinterestherecomprisesgenuine

Trametesspecies(i.e.,withstrictlyporoidhymenophores):T. versicolor,T.hirsuta,T.ochracea,T.suaveolensandT.chineseclose

toT.junipericola,T.socotrana,T.pubescensandT.villosa.Mostof

thesespecies,exceptforT.socotranaandT.villosa,arefoundin

temperateareas.

In other fungal species, such as Trametes trogii,

previ-ousdata indicatedthatconcentrations ofCu2+ _up_to₁_mM

are associated withincreased mycelial growth.25 _However,

the response toCu2+ _is_affected _by_{concentration} _because,

atextremely high doses,decreased fungal growth leadsto

decreasedlaccaseactivity.Ramsayetal.26_found_that_in

Tri-chodermavirensandRoseaclonostachys,highconcentrationsof

toxic metals,such asCu2+_, _decrease _the _carbon _utilization

rate.26_This_effect_might_occur_because_the_presence_of_Cu2+

decreasestheabilityoffungitouseresourcessuchascarbon

thatcanthereforenotbeusedforfungal nutrition.27_Some

authors have suggestedthat fungidevelop mechanismsto

protectthemagainstthetoxiceffectofions;suchmechanisms

includetheimmobilizationoftheseionsthroughthe

intra-cellularandextracellularproductionoforganicacid-chelating

compoundsandsiderophores.28

Some authors have demonstrated a close association

between proteinsecretion andenzyme activity.29 _Our_data

showed an average correlation between secreted proteins

and laccase activity,indicating that proteinconcentrations

are not a good indicator of enzyme activity; thus,laccase

activity measurementsare warranted. Itis usefultoreport

enzymeactivity inrelationtobiomasslevels(expressedas

Ug−1ofmycelium)30_because_this_parameter_can_reveal_the

trueincreaseinenzymeactivityaccountingforfungalgrowth.

(9)

laccasesecretion didnotalways followacommon pattern;

thus,generalizationsusedtopredictthebehaviorofagiven

fungusareimpeded.31

IntermsoftheeffectofCu2+ _on_laccase_production,_our

results are comparableto those obtained in other studies

onT.trogii(Levinetal.,2001),25 _Pleurotus_ostreatus5,32 _and_T.

pubescens33_;_in_these_species,_enzyme_activity_was_increased

inthepresenceofCu2+_._Enzyme_activity_was_affected_by_Cu2+

concentrations,andactivitywashigheratlower

concentra-tions,asforstrainsA,BandE.Someauthorshavedescribed

howCu2+ _interacts_with_the_ACE _{transcription} _factor-1_and

thereby induce lac gene expression.34,35 _However, _not _all

lac genes respond equally to this transcription factor and

somegenesmightberegulatedbyalternativeroutes.These

datacouldexplainthepresenceofCu2+_-inducible_and

non-inducible isoenzymes.Our study foundtwo Cu2+_-inducible

laccaseisoenzymeswithdifferentresponsestoCu2+_,_as

pre-viouslydescribed.7,11,36_All_strains_showed_an_increase_in_the

activityofbothisoenzymesinthepresenceofCu2+_._This_is_in

agreementwiththeclusteringresultsobservedinthe

phylo-geneticstudy.

MnPactivitywasaffectedinallstrainsbythepresenceof

Cu2+.Thiscouldbeduetoametalresponseelement-binding

transcriptionfactor,consistentwithpreviousdataindicating

thepossibleregulationofMnPbyextracellularconcentrations

ofmetalions, suchasCu2+ _and_Mn2+_.37–39 _In_this _context,

Alvarezetal.35_observed_an_effect_of_Cu2+_on_the_expression

ofgenes encodingligninolytic laccase(lcs) and manganese

peroxidase(mnp)inCeriporiopsissubvermispora.

Enzymaticactivity isaffected byproteins, mineralsand

chemicalsthatarepresentinculturesupernatants.40,41 _The

thermostabilityandthepHandtemperatureoptimaof

isoen-zymesshouldbedeterminedunderspeciﬁcconditions.41–45

OurdatashowedthatlaccaseactivitywasmaximalatpH3.6,

consistentwithreporteddata.46_This_value_is_somewhat_lower

thantheusualpHrangeforenzymeactivity(between4and

6).2,47

Strain B did not show signiﬁcant variation in enzyme

activityacrossthestudiedpHrange,indicatingthe

biotech-nologicalpotentialofthisstraintoperformunderacidicpH

bioprocessconditions.Similarresultshavebeenreportedby

Sathishkumaretal.48

Theoptimaltemperatureforlaccaseactivityinthe

pres-ence of Cu2+ _was ₅₅◦_C, _consistent _with _published _data.49

However,wefoundsomedifferencesinculturesupernatants

lackingCu2+_(strains_B_and_C);_these_differences_might_be_due

todifferencesinglycosylationamongtheisoenzymes.46

Thermostability is a desired property for industrial

enzymeapplicationbecauseitreducesthedemandforfresh

enzyme.50,51_Our_data_indicated_extended_laccase_stability_in

strainAattheoptimaltemperatureandpH;50%laccase

activ-ityremainedat4h,and40%activityremainedat7h.

Inconclusion, all strains were affected by the presence

ofCu2+_,_which _increased_laccase_and _MnP_activity._Laccase

increasesoccurred in thesame strain due tothe presence

oftwoisoenzymesthatrespondtoCu2+_._Strain_A_was_very

responsive to Cu2+ _and _presented _good _{thermostability,}

demonstratingitspotentialforfuturebiotechnological

appli-cations. This work constitutes a biochemical study of the

potential of newly discovered strains of WRF that secrete

laccase and their response to the presence of Cu2+_. _This

and previous studies6,7,52 _aimed _to _evaluate _the _capacity

of newly discovered native fungi to produce enzymes for

biotechnologicalapplications.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

PartoftheexperimentalworkwasfundedbytheSecretaría

de Ciencia yTecnologíade laUniversidadNacional(grants

forinnovationprojects).MIFisacareermember,EMGhasa

postdoctoralstudiesfellowship,andSSSAandMLBhave

fel-lowshipsfordoctoralstudiesfromtheConsejoNacionalde

InvestigacionesCientíﬁcasyTécnicas(CONICET-Argentina).

r

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