Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

(1)

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

Bacterial,

Fungal

and

Virus

Molecular

Biology

Cloning

and

expression

of

an

endoglucanase

gene

from

the

thermotolerant

fungus

Aspergillus

fumigatus

DBiNU-1

in

Kluyveromyces

lactis

Budsayachat

Rungrattanakasin

a

_,

_Siripong

_Premjet

b

_,

_Sudarat

_Thanonkeo

c

_,

Preekamol

Klanrit

a,d

_,

_Pornthap

_Thanonkeo

a,d,∗

a_Khon_Kaen_University,_Faculty_of_Technology,_Department_of_{Biotechnology,}_Khon_Kaen,_Thailand b_Naresuan_University,_Faculty_of_Science,_Department_of_Biology,_Phitsanulok,_Thailand

c_Mahasarakham_University,_Walai_Rukhavej_Botanical_Research_Institute,_Maha_Sarakham,_Thailand

d_Khon_Kaen_University,_Faculty_of_Technology,_Fermentation_Research_Center_for_Value_Added_Agricultural_Products,_Khon_Kaen,_Thailand

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29April2017

Accepted14October2017

Availableonline3February2018

AssociateEditor:RodrigoGalhardo

Keywords: Aspergillusfumigatus Cellulosicmaterial Genecloning Kluyveromyceslactis Thermotolerantfungus

a

b

s

t

r

a

c

t

An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was

cloned,characterizedandexpressedintheyeastKluyveromyceslactis.Thefull-lengthopen

readingframeoftheendoglucanasegenefromA.fumigatusDBiNU-1,designatedCel7,was

1383nucleotidesinlengthandencodedaproteinof460amino acidresidues.The

pre-dictedmolecular weightandtheisoelectricpointoftheA.fumigatusCel7geneproduct

were48.19kDaand5.03,respectively.AcatalyticdomainintheN-terminalregionanda

fungaltypecellulose-bindingdomain/module intheC-terminalregionweredetectedin

thepredictedpolypeptidesequences.Furthermore,asignalpeptidewith20aminoacid

residuesattheN-terminuswasalsodetectedinthededucedaminoacidsequencesofthe

endoglucanasefromA.fumigatusDBiNU-1.TheendoglucanasefromA.fumigatusDBiNU-1

wassuccessfullyexpressedinK.lactis,andthepuriﬁedrecombinantenzymeexhibitedits

maximumactivityatpH5.0and60◦C.TheenzymewasverystableinapHrangefrom4.0to

8.0andatemperaturerangefrom30to60◦_C._These_features_make_it_suitable_for_application

inthepaper,biofuel,andotherchemicalproductionindustriesthatusecellulosicmaterials.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Cellulose,themostabundantorganicandrenewablebiomass,

has been considered a good candidate for the production

ofalternativebiofuels andchemicals.1 _Cellulose_consists_of

∗ _{Corresponding}_author_at:_Department_of_{Biotechnology,}_Faculty_of_Technology,_Khon_Kaen_University,_Khon_Kaen_40002,_Thailand.

E-mail:portha@kku.ac.th(P.Thanonkeo).

alinear chainofseveral glucoseunits thatare linkedby␤

(1→4)glycosidicbonds.2–4 _It_is_one_of_the _major_structural

constituents ofplant cell wallsand iscommonly foundin

the stalks,stems,trunksand allthewoody portionsofthe

plantbody.5 _Conversion_of_cellulose_into_fermentable_sugar,

https://doi.org/10.1016/j.bjm.2017.10.001

(2)

i.e., glucose, requires the synergistic actionofmany

cellu-lolytic enzymes known as cellulases. Cellulases refer to a

multienzymecomplexcomposedofendoglucanase

(endo-1,4-␤-glucanase,EC 3.2.1.4),exoglucanase (exo-1,4-␤-glucanase,

EC3.2.1.91),andcellobiase(␤-glucosidase,EC3.2.1.21).6,7_The

enzymesarewidelyfoundinmanyorganisms,suchas

acti-nomycetes,bacteria,fungiandplants.Amongthethreemajor

groupsofcellulases,endoglucanaseisconsidered tobethe

mostimportantenzymebecauseitsactionresultsina

non-reducingendthatisnecessaryfortheactionofexoglucanase

andcellobiase.8

Thereareseveralsourcesofendoglucanasesuchas

bacte-ria,fungiandplant,7–10_but_fungal_{endoglucanase}_is_the_major

sourceofenzymeforindustrialapplications.Althoughseveral

endoglucanaseshavebeencloned,identiﬁedand

character-ized, mostefforts have focusedon the enzymes from the

ﬁlamentousfungi,suchasAspergillususamii,7 _A._kawachii,12

Trichoderma reesei,13 _A. _niger,14 _Volvariella _volvacea,15,16

Peni-cillium canescens,11 _P. _funiculosum17 _and _Verticillium _dahlia.18

Niermanetal.19_reported_that_there_are_{approximately}

eigh-teendifferentgenesthatencodeendoglucanasesintheentire

sequence oftheA. fumigatusAf293 genome.Most recently,

the endoglucanase gene belonging to the family AA9

(for-merlyknownasGH61)fromthe alkali-tolerantA.fumigatus

MKU1,whichisisolatedfrompaperandpulpindustrywastes,

hasalsobeencloned andsequenced.3 _Das_et_al.20_reported

the kinetic properties of the puriﬁed halostable

endoglu-canasefromacultureextractofA.fumigatusABK9,andthe

authorsdemonstratedthatthecalculatedmolecularweight

of the enzyme was approximately 56.3kDa. The enzyme

displayed its maximum activity at a temperature of 50◦C

and a pH value of 5.0. This enzyme is highly stable in a

pH range from 4.0 to 7.0 and at NaCl concentrations up

to3.0M. Xu et al.21 _reported _the _expression_and _secretion

offungalendoglucanaseIIandchimericcellobiohydrolaseI

in the oleaginous yeast Lipomyces starkeyi and the authors

demonstratedthattheengineeredL.starkeyiwascapableof

expressing and secreting both enzymes with high activity

towardcellulose.

Currently,Kluyveromyceslactis,anascomycetousbudding

yeastbelongingtotheendoascomycetales,isconsidered as

oneofthemostfavoredsystemsfortheexpressionof

sev-eralheterologousproteinsbecauseithasseveraladvantages

includingproteinfolding,proteinprocessingand

posttransla-tionalmodiﬁcation.IthasbeenreportedthatK.lactisproduces

andsecretesheterologousproteinsatahigherlevelthan

Sac-charomycescerevisiae.Italsosecretesfewnativeproteinsand

asecretedrecombinantproductisthereforeeasilyrecovered

andpuriﬁedfromtheculturemedium.22,23_Most_importantly,

K. lactis can be grown ininexpensive media. Furthermore,

it has been asserted as a “Generally Recognized As Safe”

microorganismbytheUSFDAandcanthusbeappliedin

sev-eralindustriessuchasfood,beverage,ﬂavor,vitaminandfeed

production.24

In the current study, the cloning and molecular

char-acterization ofthe gene encodingendoglucanase from the

thermotolerant fungus A. fumigatus DBiNU-1 were

investi-gated. In addition, the heterologous expression of the A.

fumigatusendoglucanasegeneinyeastK.lactisGG799andthe

enzymaticpropertiesofthegeneproductwerealsodescribed.

Wedemonstratedinthecurrentstudythattheendoglucanase

fromA.fumigatusDBiNU-1isathermostableenzyme.

Materials

and

methods

Strains,plasmidvectorsandcultureconditions

ThethermotolerantfungusA.fumigatusDBiNU-1usedinthis

workwasobtainedfromDepartmentofBiology,Facultyof

Sci-ence,NaresuanUniversity.Itwasisolatedfromasoilsample

collected inthenorthernpartofThailand,and was

identi-ﬁedbymorphological analysisand DNAsequencing ofthe

internaltranscribedspacer (ITS)andlarge subunit(LSU)of

rDNAregion.ItwasusedasasourceforgenomicDNA

iso-lation. Thefunguswasmaintainedonpotatodextroseagar

(PDA)and storedat4◦C.K.lactis GG799(New England

Bio-labs,US)wasusedasahostsystemforproteinexpression,

whileEscherichiacoliDH5␣wasusedasahostsystemforgene

cloning.ThepGEM-TEasy(Promega,Madison,WI,USA)and

pKLAC2vectorswereusedforgenecloninginE.coliandfor

geneexpressioninK.lactis,respectively.

GenomicDNAextraction

A.fumigatusDBiNU-1wasculturedinYPDmediumonarotary

incubatorshaker(220rpm)at30◦Cfor24h.Thefungalmycelia

were harvested byﬁltration and washed twicewithsterile

distilled water.Genomic DNAwasisolated fromthe fungal

myceliausingtheGF-1nucleicacidextractionkit(Vivantis,

USA), and the puriﬁed genomic DNA was used as a DNA

templateforgeneampliﬁcationusingthepolymerasechain

reaction(PCR)technique.

PCRampliﬁcation,cloningandnucleotidesequencingof theendoglucanasegenefromA.fumigatusDBiNU-1

Basedonthealignmentofthemultipleaminoacidsequences

oftheendoglucanasesfromA.fumigatusMKU1(AFJ54162),A.

nigerATCC10574(ADC84000)andA.oryzaeKBN616(BAA22588),

theconservedregionsattheN-terminus(GALYLSEM)and

C-terminus(IRWGDIG)werefound.Thedesignofthedegenerate

primers Cel7asper1F (5-GGNGCNYTNWSNGARATG-3) and

Cel7asper1R(5-NCCDATRTCNCCCCANCKDAT-3)werebased

onthesetworegions,andtheprimerswereusedtoamplify

apartialsequenceoftheendoglucanasegenefromA.

fumiga-tusDBiNU-1.Sincethepartialsequenceoftheendoglucanase

gene fromA.fumigatusDBiNU-1 washighlyhomologousto thatfromA.fumigatusMKU1(JQ796069),theampliﬁcationof

thefulllengthopenreadingframe(ORF)oftheendoglucanase

gene fromA. fumigatusDBiNU-1 wasperformed using

spe-ciﬁc primers Cel7full-F (5

-ATGGACTCCAAAAGAGGCGTCGT-3) and Cel7full-R (5-CTACAGACACTGAGAGTACCACGGA-3),

whichweresynthesizedbasedonthenucleotidesequences

oftheendoglucanasegenefromA.fumigatusMKU1.ThePCR

reactionmixture(25␮l)consistedof2×KODXtremeTM _PCR

buffer, 2mM dNTPs, 10pmol/␮l each forward and reverse

primer,1.0U/␮lKODXtremeTM_Hot_Start_DNA_polymerase,_and

100ng/␮ltemplateDNA,asperthemanufacturer’s

(3)

at95◦C for 3min. Thirty cycles of the ampliﬁcation

reac-tionwerethenperformedusingofthefollowingconditions:

denaturationfor1minat94◦C,annealingfor1minat50◦C

andextensionfor2minat72◦C.Aﬁnalextensionwas

per-formedat72◦Cfor7min.ThePCRproductwasvisualizedon

0.7%agarose gel,extractedand puriﬁedfrom thegelusing

theNucleoSpinExtractIIKit(Macherey-Nagel,Germany).The

RapidDNALigationandTransformationKit(Fermentas,USA)

wasusedto clonethepuriﬁed PCRproductinto thepGEM

T-easy vector and to transform the resulting plasmid into

E. coli DH5␣. Transformants were screened at 37◦C on LB

mediumcontaining 100␮g/mlampicillin. Therecombinant

plasmidDNAwasisolatedfrom theselectedtransformants

andsubjectedtoDNAsequencingbytheSangermethodusing

the MegaBACE1000automatedDNA sequencer (Pharmacia

Biotech, Sweden). The nucleotide and the deduced amino

acidsequencesoftheendoglucanasegeneproductfromA.

fumigatusDBiNU-1wereanalyzedusingGENETYX(Software

Development,Tokyo,Japan),whileahomologyanalysiswas

carriedoutusingtheFASTAandBLASTprograminthe

Gen-Bankdatabase.

Constructionoftheexpressionvectorandtransformation ofK.lactis

The ORF of the endoglucanase gene from A.

fumiga-tus DBiNU-1 was ampliﬁed by PCR using the following

primer pairs: CEL7PKLACF: 5 CGCTCGAGAAAAGAATGG

ACTCCAAAAGAGGC-3 (the underline bases

repre-sent the XhoI restriction site) and CEL7PKLACR:

5-CCGGAATTCCTACAGACACTGAGAGTACCA-3 (the

under-linebasesrepresenttheEcoRIrestrictionsite).AllPCRprimers

weresynthesizedbyIntegratedDNAtechnology(IDT,

Singa-pore).ThePCRreactionmixture(25␮l)consistedof2×KOD

XtremeTM_PCR_buffer,₂_mM_dNTPs,₁₀_pmol/_␮l_each_forward

and reverse primer, 1.0U/␮lKOD XtremeTM _Hot _Start _DNA

polymerase and 20ng/␮ltemplate DNA, as per the

manu-facturer’sinstruction (Merck,Germany). Thethermocycling

conditionsconsistedofa94◦Cstepfor2min,followedby35

cyclesofsuccessiveincubationat94◦Cfor30s,60◦Cfor1min

and72◦Cfor2min.Afterthethermocycling,aﬁnalextension

wasperformed at72◦C for7min. Toconstructthe

expres-sioncassette pKLAC2-Cel7, the puriﬁedampliﬁedfragment

wasdigestedwithXhoIand EcoRIand thenligatedintothe

XhoI/EcoRIsiteoftheK.lactisexpressionvectorpKLAC2.The

accuracyofthenucleotidesequenceofthecloned

endoglu-canasegenewasconﬁrmedbyDNAsequencingasdescribed

above.

ThetransformationoftheexpressioncassetteintoK.

lac-tisGG799wasperformedusinglithiumacetatewiththeheat

shockmethod.25_To_integrate_the_cloned_{endoglucanase}_gene

intotheLAC4locusoftheK.lactischromosome,the

expres-sioncassettepKLAC2-Cel7was linearized withBstXI before

transformation. The transformants harboring the

pKLAC2-Cel7plasmidwerescreenedandselectedat30◦Cusingyeast

carbon base (YCB)agar mediumsupplemented with 5mM

acetamide.Theselectedtransformantswerefurtheranalyzed

onYPDagarmediumcontaining1%carboxymethylcellulose

(CMC)(Fluka,Germany).

Detectionoftherecombinantendoglucanaseusing1% Congored

TheselectedtransformantsweregrowninYPGalmediumat

30◦Cinanincubatorshaker(200rpm)for24h.Thereafter,50␮l

oftheyeastculturewastransferredontotheYPDagarmedium

thatcontained1%CMCandincubatedat30◦C.Steriledistilled

waterwasusedasacontrolinthisstudy.After7daysof

incu-bation,theagarmediumwasﬂoodedwith1%Congoredand

subsequentlyincubatedatroomtemperature(RT)for15min.

Theagarmediumwasrinsedtwicewithsteriledistilledwater

andthenwashedwitha1MNaClsolutionfor15min.Theclear

zonethatappearedontheagarmediumwasmonitoredand

photographed.

SDS-polyacrylamidegelelectrophoresisandzymogram analysisoftherecombinantendoglucanase

TheselectedtransformantwasgrowninYPDliquidmedium

supplementedwith1%CMCat30◦Cinanincubatorshaker

(200rpm)for7days.Theculturesupernatantwascollected

by centrifugation at 13,000rpm and 4◦C for 15min, and

the endoglucanase activity inthe clearsupernatant (crude

enzyme)wasassayed.Theproteinconcentrationinthe

cul-ture supernatant was measured by the Bradfordmethod26

using bovine serum albumin (BSA) as the standard. For

SDS-polyacrylamidegelelectrophoresis(SDS-PAGE),thecrude

enzyme was prepared based on the method described by

ErikssonandPettersson27_and_Béguin,28_using_a_sample_buffer

containing2%SDS,0.5MTris–HCl(pH6.8),10%glyceroland

0.01%bromophenolblue.TheSDS-PAGEwascarriedoutbased

onthestandardmethoddescribedbyLi,29_using₃₀_␮g_of

pro-teinsampleanda12%polyacrylamidegel,andtheseparation

oftheproteinbandsinthegelwasvisualizedbyCoomassie

brilliantblueR-250(Sigma)staining.

The zymogram analysis was based on the method

described byGrigorevski-Lima etal.,30 _and_was _carried_out

to determine the enzymeactivity. The crude enzyme was

subjectedtoSDS-PAGEusinga12%polyacrylamidegelthat

contained1%CMCassubstrate.Afterelectrophoresis,thegel

wassoakedin2.5%(v/v)TritonX-100for2handthen

incu-batedin50mMsodiumacetatebuffer(pH5.0)for1hatRTin

ordertoremovetheSDSandrenaturetheenzyme.The

result-ing gelwas washedwith50mM sodiumacetatebuffer(pH

5.0)at45◦Cfor30min,stainedwith1%(w/v)Congoreddye

solutionatRTfor30min,andsubsequently destainedwith

1NNaCluntiltheexcessdyewasremoved.Theclearzone,

whichcorrespondedtoenzymeactivity,wasmonitoredand

documentedwithageldocumentationsystem(Bio-Rad,

Sin-gapore).

Puriﬁcationofproteinusinganaqueoustwo-phase system

The recombinant endoglucanase was puriﬁed using an

aqueous two-phase system.31 _Various _{concentrations} _of

polyethyleneglycol6000(PEG6000)anddextranT70wereused

inthissystem.Topreparethetwo-phasesystem,astock

solu-tioncomposedof20%(w/w)dextranand40%(w/w)PEGand

(4)

1.5h. Protein samplesin the top and bottomphases were

taken,and theenzymeactivity inbothphases wasfurther

determined.

Theactivity of the puriﬁed recombinant endoglucanase

wasassayedusingthemethoddescribedbyLietal.,16_using

CMCassubstrate.Oneunit(U)ofenzymeactivitywasdeﬁned

astheamountofenzymecapableofreleasing1␮molof

reduc-ingsugarfromCMCperminuteundertheassayconditions.

d-Glucosewasusedasthestandard.

EffectofpHonenzymeactivityandstability

TheeffectofpHontheactivityofthepuriﬁedrecombinant

endoglucanase was evaluated using the standard method

describedbyLietal.,16_with_different_buffers_including_sodium

acetatebuffer(pH3.0–6.0)andphosphatebuffer(pH7.0–11.0).

ThestabilityofthepuriﬁedendoglucanaseatdifferentpH

val-ueswasevaluatedbyincubatingthepuriﬁedenzymeat50◦C

for1hinthesamebuffersinthepHrangefrom 3.0to11.0

andmeasuringtheremainingenzymeactivityunderstandard

conditions.

Effectoftemperatureonenzymeactivityandstability

Theeffectoftemperatureontheactivityofthepuriﬁed

recom-binantendoglucanasewasevaluatedattheoptimumpHat

temperaturesrangingfrom30to80◦C.Thethermalstability

ofthe puriﬁedenzymewasevaluated bypreincubatingthe

enzymesolutionin50mMsodiumacetatebuffer(pH5.0)at

varioustemperatures(30–80◦C)for1h.Theremainingenzyme

activitywasassayedunderstandardconditions.

Results

Cloningandnucleotidesequencinganalysisofthe endoglucanasegenefromA.fumigatusDBiNU-1

The ORF of the gene encoding the endoglucanase in A.

fumigatus DBiNU-1 was ampliﬁed by PCR using a genomic

DNA templatethat was isolated from A. fumigatus

DBiNU-1 and speciﬁc primers whose design was based on the

nucleotide sequences of the endoglucanase gene from A.

fumigatus MKU1, as described in the Materials and

meth-ods section. A PCR product of approximately 1.3kb was

detected after agarose gel electrophoresis analysis, and it

waspuriﬁedandcloned,andtheresultingvectorwas

trans-formed into E. coli DH5␣. The recombinant plasmid DNA

thatwasisolatedfromtheselectedtransformantswasthen

subjected to DNA sequencing. As found in the present

study, the complete nucleotide sequence of the

endoglu-canasegenefromA.fumigatusDBiNU-1,designatedCel7,was

1,383 nucleotides in length and encoded a polypeptide of

460 amino acid residues (Fig. 1). The predicted molecular

weightandtheisoelectricpointoftheA.fumigatusCel7gene

product were found to be48.19kDa and 5.03,respectively.

Based on the sequence similarities in the

carbohydrate-active enzyme (CAZy) database (http://www.cazy.org/), the

deduced amino acid sequence ofthe endoglucanase gene

product from A. fumigatus DBiNU-1 was classiﬁed into

glycosidehydrolasefamily7(GH7).32–34 _These_comprise_two

domains, i.e., acatalytic domainatthe N-terminus, which

containstheglycosylhydrolasefamily7active-siteresidues

(aminoacidsatpositions22–398),andafungaltype

cellulose-bindingdomain/module(CBD/CBM)attheC-terminus(amino

acids atpositions 428–456), which is involved in substrate

binding,asobservedinmostfungalcellulases.35_The

predic-tion oftheaminoacid sequencesbythe SignalP3.0server

(http://www.cbs.dtu.dk/services/SignalP/)revealedthesignal

peptide(MDSKRGVVAAVLALLPLVSA)attheN-terminalregion

andasignalpeptidecleavagesitebetweenAla20_and_Gln21_.

The homology analysis of the deduced amino acid

sequence showed that the endoglucanase from A.

fumi-gatus DBiNU-1 shared 98%, 74%, 59%, 51%, 46% and 34%

identity with the GH7 endoglucanases from A. fumigatus

KMU1 (AFJ54162), A. terreus MS-31 (ADR78837),T. reesei M5

(ADM08177),A.oryzaeKBN616(BAA22589),A.nidulansFGSCA4

(EAA63386),andFusariumoxysporum(AAA65586),respectively

(Fig. 2). The putative catalytic amino acid residues Asp191

and Glu369 _were _found _to _be _highly _conserved _among _the

GH7endoglucanasesandaresimilartothosereportedinthe

GH12 and AA9 family.3,7 _A _{three-dimensional} _structure _of

thepredictedproteinsequenceoftheendoglucanasefromA.

fumigatusDBiNU-1waspredictedusingtheSwiss-PdbViewer (http://www/expasy.org/spdbv/), and the resultisshown in

Additionalinformationsection,Fig.1.Theproteinstructure

wasobservedatthetertiarylevel,andtwocompletelydistinct

structurescorrespondingtothecatalyticdomainandCBM1

domainweredetected.

ExpressionoftheendoglucanasegenefromA.fumigatus DBiNU-1inK.lactisGG799

The expression vector pKLAC2-Cel7 was constructed and

transformedintoK.lactisGG799,andthetransformantswere

selectedusingYCBagarmedium,asdescribedinthe

Mate-rialsandmethodssection.Theselectedtransformantswere

furtheranalyzedonYPDagarmediumcontaining1%CMC.

TransformantsharboringthepKLAC2-Cel7exhibitedaclear

zoneonagarplatesafterCongoredstaining,suggestingthat

the cloned endoglucanasegene from A.fumigatus DBiNU-1

wasexpressedintheK.lactis(Fig.3).

Thecrudeenzymeinthecell-freesupernatantwas

ana-lyzedbySDS-PAGEusinga12%polyacrylamidegel,andthe

resolved proteinswere visualizedusing Coomassiebrilliant

blue R-250 staining. As shown in Fig. 4A, a protein band

ofapproximately48kDathatcorrespondedtothepredicted

molecular weight of the endoglucanase from A. fumigatus

DBiNU-1wasdetected.Theendoglucanaseactivitywasalso

determinedbasedonthedegradationofCMCinthe

polyac-rylamide gelusingazymogramanalysis.Aproteinbandof

approximately48kDathatcoincidedwiththebandthatwas

observedintheSDS-PAGEanalysiswasdetectedona

polyac-rylamidegelafterCongoredstaining(Fig.4A),suggestingthat

thisproteinbandwastherecombinantendoglucanasefromA.

fumigatusDBiNU-1.Theendoglucanaseactivityinthecell-free

supernatantfromK.lactisharboringthepKLAC2-Cel7plasmid

was measured,and thehighestenzymeactivity wasfound

(5)

Fig.1–NucleotideanddeducedaminoacidsequencesofanintronlessendoglucanasefromthermotolerantA.fumigatus

DBiNU-1.ThesignalpeptidefromMet1_to_Ala20_is_underlined._The_conserved_amino_acid_regions,_GALYLSEM_and_IRWGDIG,

areshowninboldletters.TheputativecatalyticaminoacidresiduesAsp191_and_Glu369_are_shown_in_the_boxes.

Therewasnoendoglucanaseactivity inthecell-free

super-natantfrom thenegativecontrol(K.lactistransformedwith

pKLAC2)underthesameculturalconditions.

Puriﬁcationandcharacterizationofthepuriﬁed recombinantendoglucanase

TherecombinantendoglucanasefromtheK.lactisstrainthat

harboredpKLAC2-Cel7waspuriﬁedusingtheaqueous

two-phase system, as described in the Materials and methods

section.Amaximumenzymeactivityandspeciﬁcactivityof

1.57U/mland15.39U/mgprotein,respectively,wereachieved

usingthetwo-phasesystemthatcontained7%PEGand5%

dextran.Fig.4BshowstheSDS-PAGEanalysisofthepuriﬁed

recombinantendoglucanasethatwasobtainedfromaqueous

two-phasesystem.Mostoftheproteinbandatapproximately

48kDa,whichcorrespondedtothemonomeroftheCel7gene

productfromA.fumigatusDBiNU-1,wasobserved,whilethose

low-molecularweightproteinsthatwerecontainedinthe

cell-freesupernatantweremostlyremoved.

TheeffectofpHontheactivityandstabilityofthepuriﬁed

recombinantendoglucanasewasevaluated, andthe results

aresummarizedinFig.5A.Thepuriﬁedrecombinant

endoglu-canasedisplayeditshighestactivityatpH5.0,andmorethan

80%ofitsoriginalactivityremainedinthepHrangefrom4.0

to8.0.

Theeffect oftemperature onthe recombinant

endoglu-canase activity and stability is illustrated in Fig. 5B. The

maximalactivityofthepuriﬁedrecombinantendoglucanase

atpH5.0wasattainedatatemperatureof60◦C.Theactivity

ofthepuriﬁedenzymeremarkablydecreasedattemperatures

above60◦C.Morethan80%oftheoriginalactivityofthe

puri-ﬁedenzymeremainedinatemperaturerangefrom30to60◦C,

whereasonlyhalfoftheoriginalactivitywasdetectedat70◦C.

Discussion

Based on the whole genome sequence of A. fumigatus

Af293,18differentgenesencodingendoglucanaseshavebeen

reported.19 _They _belong _to _various _families, _such _as _GH5,

GH7,GH12,GH45,AA9(formerlyknownasGH61),andGH81,

based on their amino acid sequence similarities.

Accord-ing toanucleotidesequenceanalysis,threeendoglucanase

(6)

Fig.2–AlignmentsbetweentheA.fumigatusDBiNU-1endoglucanasesequenceandthesequencesofotherfungal endoglucanasesfromGH7.TheputativecatalyticaminoacidresiduesAsp191_and_Glu369_are_shown_in_the_boxes.

(7)

K. lactis

pKLAC2

pKLAC2-Cel7

Distilled water

K. lactis

Fig.3–TheactivityoftheendoglucanasefromA.fumigatus

DBiNU-1thatwasexpressedintheyeastK.lactis,as determinedusingYPDagarmediumcontaining1%CMC.

twofromAA9),andtwooutofthesethreeknownintronless

endoglucanasegenesarepredictedtohaveacellulose-binding

domain/module in the C-terminal region. In the current

study,agenethatencodesanintronlessendoglucanaseinA.

fumigatusDBiNU-1wascloned,anditsnucleotideand

puta-tive aminoacid sequences were analyzed using GENETYX

(Software Development,Tokyo, Japan). Theresults showed

that the complete sequence of the gene that encodes the

endoglucanaseinA.fumigatusDBiNU-1,designatedCel7,was

1383bpin lengthandencodeda polypeptideof460 amino

acid residues (Fig. 1). Based on the amino acid sequence

similarity in the CAZy database (http://www.cazy.org), the

endoglucanasefromA.fumigatusDBiNU-1wasclassiﬁedinto

theGH7family,32–34_similar_to_{endoglucanases}_from_Aspergillus

spp.suchasA.terreusMS-31,A.oryzaeKBN616,andA.nidulans

FGSCA4,andendoglucanasesfromT.reeseiM5andPenicillium

sp.C7.AccordingtothePfamtool(http://pfam.sanger.ac.uk/),

twodomains,i.e.,acatalyticdomainintheN-terminalregion

(amino acids from position 22 to 398) and a fungal type

CBD/CBMintheC-terminalregion(aminoacidsfromposition

428to456),werefoundinthededucedaminoacidsequences

ofthe A. fumigatusCel7 geneproduct. Asdemonstrated by

Sugimoto,36_the_CBD/CBM_is_important_for_the_binding_of

cel-lulose.CellulasesthatlacktheCBD/CBMexhibitedareduction

incellulosedegradation.Itshouldbenotedfromthecurrent

studythatasignalpeptidecontaining20aminoacidresidues

was detectedin theN-terminalregion, suggestingthat the

endoglucanasefrom A. fumigatusDBiNU-1 isan

extracellu-larenzymebecausethesamefeaturehasbeenfoundinan

intronlessendoglucanasefromA.fumigatusMKU1.3_In

addi-tiontoasignalpeptide,tworegions,GALYLSEMandIRWGDIG,

thatarehighlyconservedinallfungalendoglucanasesinthe

GH7familyweredetectedintheendoglucanasefromA.

fumi-gatusDBiNU-1.ThisﬁndingalsoveriﬁedthattheA.fumigatus

Cel7geneproductwasamemberofthefungalGH7family.No

putativeN-glycosylation site(N-W-T)wasfoundinthe

pre-dictedproteinsequences,suggestingthattheendoglucanase

fromA.fumigatusDBiNU-1isnotanN-glycosylatedprotein,

whichisdistinctfromthereportedcaseoftheendoglucanase

fromA.usamiiE001.7

The predicted molecular weight of the endoglucanase

fromA.fumigatusDBiNU-1,asconﬁrmedbySDS-PAGE

anal-ysis, was approximately 48kDa, which was distinct from

other endoglucanases from other Aspergillus strains, e.g.,

A. terreus M11 (25kDa),37 _A. _awamori _VTCC-F099 ₍₃₂_kDa),38

A. fumigatus KMU1 (35kDa),3 _A. _oryzae _AG1 ₍₄₅_kDa),39 _A.

usamiiE001(24kDa),7_and_A._fumigatus_ABK9₍₅₆_kDa).20_Many

kDa

A

B

72 53 43 34 26 M 1 kDa 180 130 95 72 53 43 34 26 17 M 1 2 2 3 48 48

Fig.4–SDS-PAGEandzymogramanalysisoftheendoglucanase(A)andthepuriﬁedrecombinantendoglucanase(B)fromA. fumigatusDBiNU-1.Theproteinsamples(30␮g)wereloadedonthegelandsubjectedtoelectrophoresis.(A)M,protein marker;1,thecell-freesupernatantfromK.lactisGG799;2,thecell-freesupernatantfromK.lactisharboringpKLAC2-Cel7;3, zymogramanalysisoftheendoglucanaseafterCongoredstaining.(B)M,proteinmarker;1,cruderecombinant

(8)

100 A B 90 80 Relativ e activity (%) Relativ e activity (%) 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 3 30 40 50 60 70 80 4 5 6 7 pH Optimum pH Optimum temperature Thermal stability pH stability Temperature (°C) 8 9 10 11

Fig.5–TheeffectofpH(A)andtemperature(B)onthe activityandstabilityofthepuriﬁedrecombinant

endoglucanasefromA.fumigatusDBiNU-1.Themaximum enzymeactivity(1.57U/ml)assayedunderthestandard condition(60◦C,pH5.0)wastakenasacontroland recordedas100%.

glycoside hydrolases such as GH7, GH12, GH16 and AA9

haveamodularstructurethatconsistsofacatalyticdomain

and a substrate-binding domain.40 _The _{Swiss-PdbViewer}

(http://www/expasy.org/spdbv)wasusedtopredictthe

three-dimensionalstructureofthe putativeaminoacidsequence

oftheendoglucanasefromA.fumigatusDBiNU-1(Additional

informationsection,Fig.1).TheGH7endoglucanasefromthis

thermotolerantfungusexhibitedanarrangementofcatalytic

residues and a fold that are similar to those of the

␤-1,3-glucanasesand␤-1,3-1,4-glucanasesoffamily16.41

Theaqueoustwo-phasesystemhasbeenwidelyusedto

purifyseveralenzymes.Comparedwithconventional

puriﬁca-tionmethods,thistechniqueprovidesahighyieldofrecovery

withhighlevelofenzymeactivityandstability.31_In_the

cur-rentstudy,therecombinantendoglucanasefromA.fumigatus

DBiNU-1thatwasexpressedinK.lactiswiththepKLAC2-Cel7

vectorwaspuriﬁedusingtheaqueoustwo-phasesystem.A

maximumenzymeactivityandspeciﬁcactivityof1.57U/ml

and 15.39U/mg protein, respectively, were achieved; these

valueswereapproximately2-and5-foldgreaterthanthe

cor-respondingoriginalactivities.TheoptimumpHoftheactivity

ofthepuriﬁedrecombinant endoglucanasefromA.

fumiga-tusDBiNU-1was5.0,andtheenzymewashighlystableina

pHrangefrom4.0to8.0(Fig.5A).Thepuriﬁedrecombinant

endoglucanasealsoexhibiteditshighestactivityat60◦C,and

itover80%ofitsoriginalactivityinatemperaturerangefrom

30to60◦C(Fig.5B).Theresultsofthecurrentstudywerein

goodagreementwiththosereportedformostofthe

endoglu-canasefromAspergillusstrains,suchasA.terreusDSM826,42_A.

awamoriVTCC-F099,38_A._usamii_E0017_and_A._fumigatus_ABK9.20

However,ahighthermalstability(70◦C)wasalsoreportedfor

endoglucanasesfromT.aurantiacus43_and_A._fumigatus_MKU1.3

The differences inthermal stabilityindicated the different

molecularpropertiesoftheenzymes,includingthebonding

thatstabilizesinthestructuresandtheconformationsamong

thevariousspecies.Ithasbeenreportedthatendoglucanases

withoptimalactivitiesbetween55and80◦Careconsidered

thermostable enzymes.44 _The _high _thermal _stability_of _the

endoglucanasefromA.fumigatusDBiNU-1(60◦C)makethis

enzymeagoodcandidateforapplicationinthepaperindustry

andinsacchariﬁcationprocessesfortheproductionofbiofuel

andotherchemicalsusingcellulosicmaterials.

Conﬂicts

of

interest

Theauthorsdeclarethattheyhavenoconﬂictofinterest.

Acknowledgments

Theauthors thankKhon KaenUniversityforﬁnancial

sup-portandtheFermentationResearchCenterforValueAdded

AgriculturalProducts(FerVAAPs)atKhonKaenUniversityfor

technicalsupport.

Appendix

A. Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound,

intheonlineversion,atdoi:10.1016/j.bjm.2017.10.001.

r

e

f

e

r

e

n

c

e

s

1.HongJ,WangY,KumagaiH,TamakiH.Constructionof thermotolerantyeastexpressingthermostablecellulase genes.JBiotechnol.2007;130:114–123.

2.MakutM,GodiyaE.Asurveyofcellulolyticmesophilicfungi inthesoilenvironmentofKefﬁMetropolis,NasarawaState, Nigeria.AfrJMicrobiolRes.2010;4:2191–2195.

3.MeeraB,VanithaMC,RamaniG,ManjulaA,GunasekaranP. Cloningandexpressionofanintronlessgeneencoding endoglucanasefromAspergillusfumigatusMKU1.IndianJ Biotechnol.2011;10:480–486.

4.RowellRM.HandbookofWoodChemistryandWoodComposites. NewYork,USA:CRCpress;2012.

5.ChaudharyN,QaziJI.Lignocelluloseforethanolproduction: areviewofissuesrelatingtobagasseasasourcematerial. AfrJBiotechnol.2013;10:1270–1274.

6.AdsulMG,BastawdeMG,VarmaAJ,GokhaleDV.Strain improvementofPenicilliumjanthinellumNCIM1171for increasedcellulaseproduction.BioresourTechnol. 2007;98:1467–1473.

(9)

7.ShiH,YinX,WuM,TangC,ZhangH,LiJ.Cloningand bioinformaticsanalysisofanendoglucanasegene(Aucel12A) fromAspergillususamiianditsfunctionalexpressioninPichia pastoris.JIndMicrobiolBiotechnol.2012;39:347–357.

8.WangJ,GaoG,LiY,etal.Cloning,expression,and

characterizationofathermophilicendoglucanase,AcCel12B fromAcidothermuscellulolyticus11B.IntJMolSci.

2015;16:25080–25095.

9.WeiKSC,TeohTC,KoshyP,SalmahI,ZainudinA.Cloning, expressionandcharacterizationoftheendoglucanasegene fromBacillussubtilisUMC7isolatedfromthegutofthe indigenoustermiteMacrotermesmalaccensisinEscherichiacoli. ElectronJBiotechnol.2015;18:103–109.

10.delCampilloE.Multipleendo-1,4-beta-D-glucanase (cellulase)genesinArabidopsis.CurrTopDevBiol. 1999;46:39–61.

11.ChulkinAM,LoginovDS,VavilovaEA,etal.Cloningofthe Penicilliumcanescensendo-1,4-ˇ-glucanasegeneegl3andthe characterizationoftherecombinantenzyme.ApplBiochem Microbiol.2009;45:143–149.

12.SakamotoS,TamuraG,TtoK,IshikawaT,IwanoK,Nishiya N.CloningandsequencingofcellulasecDNAfromAsperillus kawachiianditsexpressioninSaccharomycescerevisiae.Curr Genet.1995;27:435–439.

13.SandgrenM,ShawA,RoppTH,etal.TheX-raycrystal structureoftheTrichodermareeseifamily12endoglucanase3 Cel12A,at1.9 ˚Aresolution.JMolBiol.2001;308:

295–310.

14.HongJ,TamakiH,AkibaS,YamamotoK,KumagaiH.Cloning ofageneencodingahighlystableendo-ˇ-1,4-glucanase fromAspergillusnigeranditsexpressioninyeast.JBiosci Bioeng.2001;92:434–441.

15.DingSJ,GeW,BuswellJA.Secretionpuriﬁcationand characterizationofarecombinantVolvariellavolvacea endoglucanaseexpressedintheyeastPichiapastoris.Enzyme MicrobTechnol.2002;31:621–626.

16.LiJF,TangCD,ShiHL,WuMC.Cloningandoptimized expressionofaneutralendoglucanasegene(ncel5A)from VolvariellavolvaceaWX32inPichiapastoris.JBiosciBioeng. 2011;111:537–540.

17.CastroAM,CaryalhoMLA,LeiteSGF,PereiraNJr.Cellulases fromPenicilliumfuniculosum:production,propertiesand applicationtocellulosehydrolysis.JIndMicrobiolBiotechnol. 2010;37:151–158.

18.EboigbeL,TzimaAK,PaplomatasEJ,TypasMA.Theroleof the␤-1,6-endoglucanasegenevegBinphysiologyand virulenceofVerticilliumdahliae.PhytopatholMediterr. 2014;53:94–107.

19.NiermanWC,PainA,AndersonMJ,etal.Genomicsequence ofthepathogenicandallergenicﬁlamentousfungus Aspergillusfumigatus.Nature.2005;438:1151–1156.

20.DasA,JanaA,PaulT,etal.Thermodynamicsandkinetic propertiesofhalostableendoglucanasefromAspergillus fumigatusABK9.JBasicMicrobiol.2014;54:S142–S151.

21.XuQ,KnoshaugEP,WangW,etal.Expressionandsecretion offungalendoglucanaseIIandchimericcellobiohydrolaseI intheoleaginousyeastLipomycesstarkeyi.MicrobCellFact. 2017;16:126.

22.VanderVlugt-BergmansC,VanOoyenA.Expressioncloning inKluyveromyceslactis.BiotechnolTech.1999;13:87–92.

23.SchaffrathR,BreunigKD.Geneticsandmolecularphysiology oftheyeastKluyveromyceslactis.FungalGenetBiol.

2000;30:173–190.

24.BonekampJF,OosteromJ.OnthesafetyofKluyveromyces lactis–areview.ApplMicrobiolBiotechnol.1994;41:1–3.

25.ItoH,FukudaY,MurataK,KimuraA.Transformationof intactyeastcellstreatedwithalkalications.JBacteriol. 1983;153:163–168.

26.BradfordMM.Arapidandsensitivemethodforthe

quantitationofmicrogramquantitiesofproteinutilizingthe principleofprotein-dyebinding.AnalBiochem.

1976;72:248–254.

27.ErikssonKE,PetterssonB.Azymogramtechniqueforthe detectionofcarbohydrases.AnalBiochem.1973;56:618–620.

28.BéguinP.Detectionofcellulaseactivityinpolyacrylamide gelsusingCongored-stainedagarreplicas.AnalBiochem. 1983;131:333–336.

29.LiSFY.CapillaryElectrophoresis:PrinciplesPracticeand Applications.Singapore:Elsevier;1992.

30.Grigorevski-LimaA,DaVinhaF,SouzaD,etal.Aspergillus fumigatusthermophilicandacidophilicendoglucanases.Appl BiochemBiotechnol.2009;155:18–26.

31.AsenjoJA,AndrewsBA.Aqueoustwo-phasesystemsfor proteinseparation:aperspective.JChromatogrA. 2011;1218:8826–8835.

32.NaumoffDG.Hierarchicalclassiﬁcationofglycoside hydrolases.Biochemistry.2011;76:622–635.

33.LombardV,GolacondaRamuluH,DrulaE,CoutinhoPM, HenrissatB.Thecarbohydrate-activeenzymesdatabase (CAZy)in2013.NucleicAcidsRes.2014;42:490–495.

34.TerraponN,LombardV,GilbertHJ,HenrissatB.Automatic predictionofpolysaccharideutilizationlociinBacteroidetes species.Bioinformatics.2015;31:647–655.

35.Marchler-BauerA,DerbyshireMK,GonzalesNR,etal.CDD: NCBI’sconserveddomaindatabase.NucleicAcidsRes. 2014;34:222–226.

36.SugimotoN(Ph.D.thesis)FunctionalAnalysisandApplication ofFungalCellulose-BindingDomains.Japan:Shinshu

University;2012.

37.GaoJ,WengH,XiY,ZhuD,HanS.Puriﬁcationand characterizationofanovelendo-ˇ-1,4-glucanasefromthe thermoacidophilicAspergillusterreus.BiotechnolLett. 2008;30:323–327.

38.NguyenVT,QuyenDT.Puriﬁcationandpropertiesofanovel thermoactiveendoglucanasefromAspergillusawamori VTCC-F099.AustJBasicApplSci.2010;4:6211–6216.

39.YoussefGA.Physiologicalstudiesofcellulasecomplex enzymesofAspergillusoryzaeandcharacterizationof carboxymethylcellulase.AfrJMicrobiolRes.2011;5:1311–1321.

40.DaviesG,HenrissatB.Structuresandmechanismsof glycosylhydrolases.Structure.1995;3:853–859.

41.DivneC,StåhlbergJ,ReinikainenT,etal.The

three-dimensionalcrystalstructureofthecatalyticcoreof cellobiohydrolaseIfromTrichodermareesei.Science. 1994;265:524–528.

42.ElshafeiAM,HassanMM,HarounBM,Abdel-FatahOM,Atta HM,OthmanAM.Puriﬁcationandpropertiesofan

endoglucanaseofAspergillusterreusDSM826.JBasicMicrobiol. 2009;49:426–432.

43.VoutilainenSP,PuranenT,Siika-ahoM,etal.Cloning, expression,andcharacterizationofnovelthermostable family7cellobiohydrolases.BiotechnolBioeng.

2008;101:515–528.

44.MaheshwariR,BharadwajG,BhatMK.Thermophilicfungi: theirphysiologyandenzymes.MicrobiolMolBiolR. 2000;64:461–488.