Multi-enzyme complex of white rot fungi in saccharification of lignocellulosic material

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

Short

communication

Multi-enzyme

complex

of

white

rot

fungi

in

saccharification

of

lignocellulosic

material

Wilton

Soares

Cardoso

_,

_Paula

_Viana

_Queiroz

_,

_Gabriella

_Peterlini

_Tavares

_,

Fernando

Almeida

Santos

_,

_Filippe

_Elias

_de

_Freitas

_Soares

_,

_Maria

_Catarina

_Megumi

Kasuya

_,

_José

_Humberto

_de

_Queiroz

a_{InstitutoFederaldoEspíritoSanto,VendaNovadoImigrante,ES,Brazil} b_{UniversidadeFederaldeVic¸osa,DepartamentodeQuímica,Vic¸osa,MG,Brazil} c_{UniversidadeFederaldeVic¸osa,DepartamentodeBioquímica,Vic¸osa,MG,Brazil}

d_{UniversidadeEstadualdoRioGrandedoSul,DepartamentodeBioenergia,PortoAlegre,RS,Brazil} e_{UniversidadeFederaldeVic¸osa,DepartamentodeMicrobiologia,Vic¸osa,MG,Brazil}

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Received8February2017 Accepted18May2018

Availableonline14August2018 AssociateEditor:SolangeI. Mussatto Keywords: Enzymes Cellulases Fungus Hydrolysis

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The multi-enzymecomplex(crudeextract)ofwhiterotfungiPleurotusostreatus, Pleuro-tuseryngii,Trametesversicolor,PycnosporussanguineusandPhanerochaetechrysosporiumwere characterized,evaluatedinthehydrolysisofpretreatedpulpsofsorghumstrawand com-paredefficiencywithcommercialenzyme.Mostfungicomplexeshadbetterhydrolysisrates comparedwithpurifiedcommercialenzyme.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Enzymesextractsfromdifferentfungihavebeen astrategy usedinsaccharificationoflignocellulosicbiomass,blending(2 ormoreextractsofdifferentfungi)oronlyextract.Thecrude enzymeextractsofferslowcost,noactivitiesarelostin con-centration/purificationprocesses,awidespectrumofenzyme activitiesismaintainedandsynergyamongenzymes.1–6

∗ _{Correspondingauthor.}

E-mail:wilton.cardoso@ifes.edu.br(W.S.Cardoso).

Thisstudyproduced,bysolid-statefermentation(SSF)of foragesorghumstraw,thecrudeextracts(calledmulti-enzyme complex)of5differentfungi.Thecomplexesobtainedofeach of the fungi were characterized to the apparent activities of cellulases and were evaluated forenzymatic saccharifi-cationofowninnaturasorghumstraw(not pretreated)and

https://doi.org/10.1016/j.bjm.2018.05.006

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

sorghumpretreatedstrawpulps,andtheresultscomparedto thehydrolysiscarriedoutbyacommercialcellulasecomplex. Wereevaluatedthepotentialofcrudeenzymaticextracts ofwhite rot fungi: Pleurotus ostreatus PLO06, Pleurotus eryn-giiPLE04,TrametesversicolorTRAM01,Pycnosporussanguineus

PYC02and Phanerochaetechrysosporium PC and,obtainedby SSFonstrawforagesorghum.Fungiarefromthecollectionof theDepartmentofMicrobiology,FederalUniversityofVic¸osa, Vic¸osa,MinasGerais–Brazil.

ToSSFwereusedstrawforagesorghumBRS655(stemand leaveswithoutthepaniclewiththegrain)cultivardeveloped byEMBRAPAMaizeandSorghumandcultivatedinthecityof SeteLagoas.Sorghumwascutwith120daysofplantingand sundried,groundandstoredinadryplaceawayfromlight andmoisture.

Thesorghumstraw wasmoistenedforfinalhumidityof 70%.100gofthepreparedsubstratewereplacedin polypropy-lenefilterbagsandsealedwithadhesivetapeforsubsequent autoclavingat121◦Cfor60min.Aftercooledtoroom temper-ature,eachbagwithsubstratereceivedtwodiscsofmycelia, withadiameterof2inches,ofeach funguspreviously cul-turedinPetridishofBDAfor7daysat30◦C.Thebagswere inoculatedinalaminarflowcabinetandincubatedinaBOD at28◦Cuntilcompletecolonizationofthesubstrate. Monitor-ingofthemycelialgrowthwascarriedoutvisually.Afterthe totalsubstratecolonizationbyfungi,20daysafterinoculation wereobtainedthecrudeextracts(ormulti-enzymecomplex). To obtain the enzymeextract, 5gsamples of substrate wereplacedin250mLErlenmeyerflaskscontaining50mLof sodiumcitratebuffer(50mMpH4.8)andshakenat150rpm for2hat5◦C.Thenfilteredthroughasieveandplacedin2mL Eppendorftubesfollowingcentrifugationat12,000×gat5◦C. ThesupernatantwastransferredtoanotherEppendorftube, andsubsequentlyidentifiedwiththefungusandincubation time,sealedandstoredat−18◦_C.

The reagents used in this study were purchased from SigmaChemicalscompaniesorVetecchemistrywith analyti-calgrade.

TotalcellulaseactivityorFilterpaperactivity(FPase)was determinedessentiallyaccordingtotheIUPAC7_{instructions,}

andtheliberatedreducingsugarswereestimatedbytheDNS method.8_{FPaseactivitycorrespondsto1␮Mofreducing}

sug-arsasglucoseequivalentsliberatedperminundertheassay conditions.

Endoglucanase activity (carboxymethylcellulase, EC 3.2.1.4) or carboxymethycellulase (CMCase) activity was estimatedbyadding250␮Loftheenzymecomplexin1mLof 1%solutionofcarboxymethylcellulosein0.05Mcitratebuffer, pH4.8andincubatedat50◦Cfor30min.OneCMCaseunitis theamountofenzymenecessarytoproduce1␮Mreducing sugar as glucose equivalents per min under the standard assayconditions.

Exoglucanaseactivity(EC3.2.1.91)orAVICELaseconsisted of adding 250␮L ofcrude enzymecomplex in 1mL of 1% solutionofmicrocrystallinecellulose(Avicel)in0.05Mcitrate buffer,pH4.8andincubatedat50◦Cfor30min.Periodically, theenzyme-substratesystemwasstirredinordertomaintain thepulpinsuspension.OneAVICELaseunitistheamountof enzymenecessarytoproduce1␮Mreducingsugarasglucose equivalentsperminunderthestandardassayconditions.

Xylanase activity (endo-1,4-␤-xylanase, EC 3.2.1.8) was determined inthemixtureof1mLoftheenzymecomplex, 1mLofxylansolution(1%xylanbirchhood–SIGMA)incitrate buffer0,05M,pH4.8andincubatedat50◦Cfor30min. Peri-odically,theenzyme-substratesystemwasstirredinorderto maintainxylansuspension.OneXylanaseunitistheamount ofenzymenecessarytoproduce1␮Mreducingsugaras glu-coseequivalentsperminunderthestandardassayconditions. The liberated reducing sugars were estimatedby the DNS method.7

The␤-glucosidase(EC3.2.1.21)activitywasdeterminedby incubating1mLofp-nitrophenyl-␤-d-glucopyranoside(PNPG) substrate0.005M,0.05Mcitratebuffer,pH4.8with100␮Lof theenzymecomplex(crudeextract),for15minat50◦C.The reactionwasstoppedbyadding2.0mLof1.0Msodium bicar-bonateandabsorbancewasmeasuredat410nm.Theunitof ␤-glucosidaseactivitywasdefinedastheamountofenzyme capableofreleasing1␮molofp-nitrophenolperminuteunder thetestconditions.

Laccase activity was determined by the oxidation of 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; SIGMA, St.Louis,USA)at37◦CaccordingtoBuswell etal.9

Thereactionmixture(1mL)contained600␮Lenzymeextract, 300␮LsodiumacetatebufferpH5.0(0.1M)and100␮LABTS solution(1mM).Oxidationwasfollowedviatheincreasein absorbanceat420nm( 420=36.000M−1cm−1).Oneunitof enzymeactivitywasdefinedastheamountofenzyme oxidiz-ing1␮molofABTSperminute.

QuantificationofproteinusedthemethodofBradford.10

TheapparentactivityofenzymesFPase(TotalCellulase) AVICELase,CMAaseand␤-glucosidasewerecharacterizedas topHandtemperature,aswellasthethermostabilityof max-imal enzyme activity in the temperature through tests to determineactivity ofthose enzymewithvaryingpHofthe bufferorthereactiontemperature.

ThepHrangedfrom3to8.Thebuffersystemsusedwere 50mMcitratebuffer(pH3.0–6.0)and50mMphosphatebuffer (pH6.0–8.0).Theincubationtemperatureforthemeasurement ofenzymeactivityrangedfrom30to80◦C.Toestimatethe thermostability,theenzymaticcomplex(samples)wasstored intheapparentoptimumtemperature(resultsoftemperature characterization) ofeach enzymeinthe extractcrude, and thenmadetomeasuretheresidualactivityoftheenzyme12 in12hfor48h.

The saccharification experiments were conducted in Erlemeyers 125mL in shaker (Tecnal – TE-421) stirred at 120rpmat50◦C.0.5mLsampleswerecollectedevery6hto 24h,andafter1212-hto72h,andeachsamplewasheatedat 100◦Cfor5mintoinactivatetheenzymes,centrifuged and subsequently was determined the concentration of reduc-ing sugars and glucose. Saccharification was performed in naturasorghumstraw(notpre-treated)andpulpsofthe for-agesorghumobtainedpretreatmentsasCardosoetal.11 _For

comparisonwiththe commercial enzyme(enzymatic com-plexGenencorMultifectGC)wereconductedsaccharification ofthepulpobtainedpretreatmentacid/delignifiedofthe for-agesorghumasCardosoetal.11

LAP008protocolusedasenzymesaccharificationmethod byNREL,12_{suggestsanenzymeloadintheorderof25FPUg}−1

Table1–Apparentenzymaticactivities(Ug−1dm)oftheextractsofsorghumstraw,fermentedbywhiterotfungi,after20

days,with70%humidityandincubatedat28◦C.Uistheamountofenzymerequiredtogenerate␮molproductsper

minute.

Fungi Celullasetotal (FPase)(Ug−1) AVICELase (Ug−1) CMCase (Ug−1) ␤-Glucosidase (Ug−1) Xylanase (Ug−1) Laccase (Ug−1) Proteinmgg−1 colonized substrate P.sanguineusPYC02 0.80±0.05 0.88±0.08 2.03±0.18 0.38±0.03 8.43±0.00 14.33±0.57 1.33±0.13 P.ostreatusPLO06 1.32±0.03 4.12±0.02 2.25±0.07 0.18±0.01 1.33±0.00 5.65±0.42 1.67±0.14 P.eryngiiPLE04 0.65±0.01 3.12±0.02 1.43±0.05 0.23±0.03 1.80±0.10 6.58±0.68 2.30±0.20 P.chrysosporiumPC 1.13±0.04 0.65±0.04 4.03±0.10 1.08±0.04 2.37±0.15 0.70±0.08 1.96±0.10 T.versicolorTRAM01 1.02±0.05 1.43±0.07 2.25±0.10 1.88±0.05 8.03±0.01 9.87±0.53 1.65±0.12

itwasnotutilizedextractspurified,thatismoreconcentrated

inrelationU/mL,therewerechangesinloadenzymestoabout

8FPUg−1biomass(drybasis)foreachfungusandthe

com-mercialenzyme.Theloadofsubstratewas0.3gofmaterial

lignocellulosicfor1%w/w(drybiomass)ineachflask(straw

offoragesorghuminnatura,pulpacid,pulpdelignifiedand

pulpacid/delignified,allthepulpsobtainedbypretreatment

offoragesorghum)11_{foreachsaccharificationexperiments.}

Valuesareexpressedasmeans±S.D.(StandardDeviation). Comparingthepercentagesofenzymaticsaccharificationacid pulp/delignifiedbyenzymeextractsoffungusandthe com-mercialenzymeitwasused.

The apparent total cellulase activities (FPase), CMCase, AVICELaseand ␤-glucosidase, xylanase and laccase in the crudeextractofwhite rotfungi,after20 daysof fermenta-tionofsorghumstrawwith70%humidityatatemperatureof 28◦CareshowninTable1.

Thesolidstatefermentationofsorghumstrawwascapable ofgeneratingcomplexmulticellulases,xylanaseandlaccase, withvariationsofvaluesforeachfungus.

ThecharacterizationoftheapparentCellulaseTotal Activ-ity, CMCase, AVICELase and ␤-glucosidase from white rot fungi,inrelationtopH,temperatureandthermostabilityare showninTable2.

Fig.1showsthepercentageofhydrolysisofthesorghumin naturaandpretreatpulpsbymulti-enzymecomplexes(crude extracts)ofwhiterotfungi,generatedbySSFofthesorghum straw.

Tocomparethemulti-enzymecomplexesofthefungiwith a complex ofcommercial enzymes wasperformed hydrol-ysisofacid/delignifiedpulp(higherpercentageofcellulose, lessthan1%ofligninandhemicellulose).11 _{Forcommercial}

enzyme were kept the same hydrolysis conditions applied to the extracts of fungi, the commercial enzyme (about 273 FPU/mL) was diluted 130-fold to load of enzymes of the 8.66FPUg−1 of cellulosee and 8.40FPUg−1 ofbiomass forpretreatmentacid/delignifiedpulp(alteredprotocol LAP 00812_{). Fig. 2 shows the results of saccharification of the}

acid/delignifiedpulpbymulti-enzymecomplexoffivefungi andcommercialenzyme.

Thepresentstudywasnotcarriedoutanyenrichmentor pretreatment ofthe substrate,only sterilization,controlof theincubationtemperature(28◦C)andhumidity(70%).The low yieldin cellulases may be associatedwith the insolu-ble substrate (straw)and lownitrogenlevels intheculture medium.OtherauthorshavereportedFPasevalueswellabove obtainedin thiswork. Inresearch conductedbyElisashvili andKachlishvili,13_{thefungusP.ostreatusreachedvaluesnear}

Table2–Thecharacterizationoftheapparenttotalcellulaseactivity,CMCase,AVICELaseand␤-glucosidaseofwhiterot

fungiinforagesorghumstrawSSF.a_{Theapparentthermostabilitywasevaluatedattheapparentoptimumtemperature}

ofeachenzyme.

Activitiesofcrudeextracts Apparentoptimalparameter Fungi

PC PYC TRAM01 PLE04 PLO06

Cellulasetotal (FPAse) pH 5.0 5.0 4.0 7.0 7.0 Temperature 60◦C 70◦C 50◦C 50◦C 60◦C Thermostabilitya _{40% 35% 35% 20% 10%} CMCase pH 4.0 4.0 4.0 7.0 5.0 Temperature 50◦_{C 50}◦_{C 50}◦_{C 50}◦_{C 60}◦_C Thermostabilitya _{30% 50% 60% 0% 0%} Avicelase pH 6.0 6.0 6.0 6.0 6.0 Temperature 50◦C 50◦C 50◦C 50◦C 50◦C Thermostabilitya _{20% 0% 25% 0% 0%} ␤-Glucosidase pH 7.0 7.0 5.0 5.0 5.0 Temperature 50◦C 70◦C 60◦C 40◦C 40◦C Thermostabilitya _{40% 40% 50% 80% 85%}

0% 10% 20% 30% 40% 50% 60%

"In natura" sorghum straw (not

pre-treated) Acid Pretreatment Pulp Alkali Pretreatment Pulp Acid/delignification Pretreatment Pulp % h y dr ol yz e d bi omass PYC02 PLE04 PLO06 PC TRAM01

Fig.1–Saccharificationofforagesorghumstraw“innatura”(withoutpre-treatment)andofthepulpsobtainedfrom pretreatmentaccordingtoCardosoetal.,11_{bycrudeextractsoffungi,P.chrysosporiumPC,P.sanguineusPYC02,T.versicolor} TRAM01,P.eryngiiPLE04eP.ostreatusPLO06,producedbySSFofforagesorghumstraw.

0% 4% 8% 12% 16% 20% 24% 28% 32% 36% 40%

PYC02 PLE04 PLO06 PC TRAM01 Commercial enzyme % h y dr ol y z e d c e llul os e pu lp pr e tr e a te d

Fig.2–Saccharificationofthepulpobtainedfromstrawforagesorghumpretreatedbyacidanddelignification,byfungusof multi-enzymecomplexes,P.chrysosporiumPC,P.sanguineusPYC02,T.versicolorTRAM01,P.eryngiiPLE04andP.ostreatus PLO06,producedbySSF,andcommercialenzyme(MultifectGC).

12.0Ug−1 forFPase and CMCase activity between6.25 and 325Ug−1, inwheatstrawfermentationenrichedwithyeast extract.

The productionxylanase by solid-state fermentation of sorghumstrawwasthemostsignificantamongthehydrolytic enzymes.

Inapparentactivityoflaccase,thehighlightwasagainthe

P.sanguineusPYC02withthevalueof14.33Ug−1(dm).Thereis anincreasingtrendinemploymentoflaccasein biotechno-logicalprocesses,14,15_{andP.sanguineushasbeenusedinKraft}

bleachingeffluent16 _{anddegradation differentdyes.}17

How-ever,thevalueobtainedbyP.sanguineusPYC02waslowerthan thatreportedbyotherstrainsofP.sanguineus,asinthework ofVikineswaryetal.,18_{wheretheproductionoflaccaseinSSF}

organicresiduesreached48.7Ug−1(dm).

Theextracellularproteinvaluesoffungitestedinthisstudy

(Table1)werelowerthanthosereportedinotherstudies,19,20

possiblyduetolackofenrichmentwithnitrogenandalsothe factthatthebiomassusedislowproteinlevel.

Resultsofthethermostabilityof␤-glucosidases,theone that deserves to be highlighted was of the P. sanguineus

PYC02 with optimum temperature of 70◦C and thermal stability(70◦C)for48hwithresidualactivityabout30%. Ther-mostablecellulasesareconsideredidealforbiotechnological applications.21 _{Another ␤-glucosidase, of the T. versicolor}

TRAM01,incubatedat60◦Cremainedover50%activityafter 48h.

The alkali pulp reachedvalues close to50% of saccha-rified biomass, followed by acid/delignified pulp with the percentage of 36.2% after 72h of saccharification (Fig. 1). Because thesematerialsmore easilydigestible due to pre-treatments, this wasexpected.Itwas alsoexpectedhigher proportionofhydrolysisoftheacid/delignifiedpulpby con-taininglowercontentofligninandhemicelluloseandhigher cellulose.

Siqueira20 reportedthatinuntreatedbagasse hydrolysis bycrudeextractofAspergillusawamoriachievedisabout30% degradationafter24hofincubation,andabout60%after96h, aresultaboveobtainedinthiswork,whichreacheda maxi-mumof20%after72h.

The fungiPleurotus PLE04and PLO06 highlightedby the lower yield inthe saccharificationwithincreasedcellulose contentofthepretreatedmaterials.Suchfactcanbelinked to ␤-glucosidase enzyme load of fungi PLE04 and PLO06

(Table1),whicharesmallercomparedtootherfungi.Thefinal

accumulationofcellobioseorglucosewillinhibitthe cellu-lose hydrolysisreactions,and cellobioseisamoreeffective inhibitorthanglucose.22,23_{Nohydrolysisofcellobiosedueto}

thelowlevelof␤-glucosidasecanreachlowlevelsof sacchar-ification.

TheenzymaticcomplexoftheP.chrysosporiumPCshowed thehighestresultsinthehydrolysisofthestrawinnaturaand ofthethreepulps.Highlightedforalkalipulpwith50%ofthe biomasshydrolyzed.InMayrink24_{theextractofTrichoderma}

spp.C012wasobtainedinabout50%hydrolysisoftheAVICEL (5FPUg−1)atjustover10hofreaction.Asimilarresulttothe PC,althoughtheloadoftheP.chrysosporiumPCwasslightly higher(8FPUg−1).

AscanbeobservedinFig.2,mostofthefungiobtained significantlybetterresultsthanthecommercialenzymethat onlyreached3%,inrelationacellulosematerial.Itmustbe rememberedthatthiscomplexofcommercialenzymes(only cellulases)wasdiluted130times.Undernormalconditionsthe samecommercialenzymes(load50FPUg−1biomass)achieved 95%yieldinthehydrolysisofacid/delignifiedpulpofsorghum straw11_{.Falkoskietal.}25 _{comparedtheextractproduced by}

fungusChrysoporthecubensiswithacommercialenzyme prod-uctandfoundthattheproducedextractwasmoreefficient forhydrolyzingalkalipretreatedsugarcanebagasseperFPU ofenzymeapplied.

Attheendispossibletoevaluatethattheuseofenzymatic complexesobtainedfromthesefungihavethe potentialto saccharification,andtheconcentrationoftheseextractscan furtherincreaseyields.

Funding

This study was funded with financial aid by Fundac¸ão de AmparoaPesquisadoEstadodeMinasGerais(FAPEMIG)and schorlarshipbyConselhoNacionaldeDesenvolvimento Cien-tíficoeTecnológico(CNPq).

Conflict

of

interest

Authorsdeclarethathavenoconflictofinterest.

Acknowledgments

TheauthorsacknowledgeIFES,FAPEMIGandCNPqfor finan-cialsupport.

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