• Nenhum resultado encontrado

Isolation and characterization of a novel endo-β-1,4-glucanase from a metagenomic library of the black-goat rumen

N/A
N/A
Protected

Academic year: 2021

Share "Isolation and characterization of a novel endo-β-1,4-glucanase from a metagenomic library of the black-goat rumen"

Copied!
8
0
0

Texto

(1)

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

Biotechnology

and

Industrial

Microbiology

Isolation

and

characterization

of

a

novel

endo-

␤-1,4-glucanase

from

a

metagenomic

library

of

the

black-goat

rumen

Yun-Hee

Song

a,1

,

Kyung-Tai

Lee

b,1

,

Jin-Young

Baek

a

,

Min-Ju

Kim

a

,

Mi-Ra

Kwon

b

,

Young-Joo

Kim

b

,

Mi-Rim

Park

b

,

Haesu

Ko

b

,

Jin-Sung

Lee

c,∗

,

Keun-Sung

Kim

a,∗ aDepartmentofFoodScienceandTechnology,Chung-AngUniversity,Ansung456-756,SouthKorea

bAnimalGenomicsandBioinformaticsDivision,NationalInstituteofAnimalScience,RuralDevelopmentAdministration,Wanju,South

Korea

cDepartmentofBiologicalSciences,KyonggiUniversity,Suwon,SouthKorea

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10August2016

Accepted1March2017

Availableonline26June2017

AssociateEditor:SolangeI.

Mussatto

Keywords:

Blackgoats

Endo-␤-1,4-glucanase

Glycosylhydrolasefamily5(GH5)

Metagenomiclibrary

Rumen

a

b

s

t

r

a

c

t

The varioustypesoflignocellulosicbiomassfoundinplants comprisethe most

abun-dantrenewablebioresourcesonEarth.Inthisstudy,theruminalmicrobialecosystemof

blackgoatswasexploredbecauseoftheirstrongabilitytodigestlignocellulosicforage.A

metagenomicfosmidlibrarycontaining115,200cloneswaspreparedfromtheblack-goat

rumenandscreenedforanovelcellulolyticenzyme.TheKG35gene,containinganovel

glycosylhydrolasefamily5cellulasedomain,wasisolatedandfunctionallycharacterized.

Thenovelglycosylhydrolasefamily5cellulasegeneiscomposedofa963-bpopen

read-ingframeencodingaproteinof320aminoacidresidues(35.1kDa).Thededucedamino

acidsequenceshowedthehighestsequenceidentity(58%)forsequencesfromthe

glyco-sylhydrolasefamily5cellulases.Thenovelglycosylhydrolasefamily5cellulasegenewas

overexpressedinEscherichiacoli.Substratespecificityanalysisrevealedthatthisrecombinant

glycosylhydrolasefamily5cellulasefunctionsasanendo-␤-1,4-glucanase.The

recombi-nantKG35endo-␤-1,4-glucanaseshowedoptimalactivitywithintherangeof30–50◦Cata

pHof6–7.ThethermostabilitywasretainedandthepHwasstableintherangeof30–50◦C

atapHof5–7.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

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

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

Introduction

Celluloseisamajorpolysaccharidecompoundinthe plant

cellwallandiscomposedofrepeatingcellobioseunitslinked

via␤-1,4-glycosidic bonds. It isone of the most abundant

Correspondingauthors.

E-mails:lejis@daum.net(J.Lee),keunsung@cau.ac.kr(K.Kim).

1 Theseauthorscontributedequallytothiswork.

renewablebioresourcesinnature.1Theenzymecellulasecan

becategorizedintothreemajorclassesbasedonitscatalytic

action:endo-␤-1,4-glucanases(EC3.2.1.4),cellobiohydrolases

(EC 3.2.1.91), or ␤-glucosidases (EC 3.2.1.21). Endo-

␤-1,4-glucanases randomly attack internal amorphous sites in

http://dx.doi.org/10.1016/j.bjm.2017.03.006

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC

(2)

cellulosepolymers,generatingnewchainends.

Cellobiohy-drolases remove cellobiose from reducing or non-reducing

ends of cellulose polymers. ␤-Glucosidases hydrolyze

cel-lodextrins and cellobioses to glucoses.2 Cellulases have

generatedcommercial interestinvarioussectors,including

the food, energy, pulp,and textile industries.3 Amongthe

three typesofcellulases, endo-␤-1,4-glucanase can release

smallercellulosefragmentsofrandomlength.4Consequently,

endo-␤-1,4-glucanasehasabiotechnologicalpotentialin

vari-ousindustrialapplications.Thisenzymehasbeenindustrially

appliedinbiomasswastemanagement,pulpandpaper

deink-ing,andtextile biopolishing.5 Inaddition, this enzymecan

bepracticallyusedtoproducebetteranimalfeeds,improve

beerbrewing,decreasetheviscosityof␤-glucansolutions,and

improvebiofinishinginthetextileindustry.5

Cellulasescanbeproducedusingawiderangeof

microor-ganisms,plants,andanimals.Symbioticmicroorganismsin

therumenofherbivorescanhydrolyzeandfermentcellulosic

polymers,therebyallowingthehosttoobtainenergyfromthe

indigestiblepolymers.6Therefore,rumenmicrobial

commu-nitiesinruminantsareattractivesourcesofcellulasesbecause

thesecommunitieshaveadaptedtoutilizationof

lignocellu-losicplantbiomass.6–9

Although the ruminal microbial ecosystem is highly

diverse, the majority of rumen microorganisms are

unculturable.6Itisestimatedthatlessthan1%ofmicrobial

species in nature have ever been cultured.10 A

culture-independentmethodhas beendevelopedto overcomethis

problem. This approach involves isolating metagenomic

DNA from the environment and cloning this DNA into a

vector to generate a metagenomic library. The library is

subsequentlyscreenedforgenesofinterestviathedetection

of genotypic or phenotypic biomarkers using DNA–DNA

hybridization,polymerasechainreaction(PCR),orenzymatic

assaytechniques.11

The Korean black goat is a grazing herbivore with a

dietary preference for herbaceous and woody dicots, such

asforbs, shrubleaves,and stems.12 Blackgoats are

classi-fiedassmall ruminants,and theirforegutdigestion allows

feedparticlestomovemorerapidlythroughthealimentary

tract,promotinghigherherbintakeincomparisonwithother

larger herbivores.13 Therefore, the objectives of this study

weretoisolateandfunctionallycharacterizeanovelcellulase

metagenomicallyderivedfrom therumencontentsofblack

goats.

Materials

and

methods

Constructionandfunctionalscreeningofametagenomic fosmidlibrary

Allanimalstudiesand standardoperatingprocedureswere

approved by the Institutional Animal Care and Use

Com-mittee(IACUC) oftheNational InstituteofAnimal Science,

RuralDevelopmentAdministration,Suwon,Korea(No.

2009-007,D-grade,surgery).Three18-month-oldKoreanblackgoats

wereraisedattheNationalInstituteofAnimalScienceand

were freely fed rice straw and mineral supplements 30d

priortotheexperimenttomaximizecellulolyticadaptation

ofmicroorganismsintheir rumens.Rumencontentsofthe

three blackgoats were collected immediately after

slaugh-ter.MetagenomicDNAwasextractedfromthecontentsusing

hexadecyltrimethylammoniumbromideandsodiumdodecyl

sulfate (CTAB-SDS).14 DNA fragments ranging from 10 to

50kbwereobtainedbypartialdigestionwiththerestriction

enzyme HindIIIto constructa metagenomic fosmid library

bymeansoftheCopyControlTMFosmidLibraryConstruction

Kit (Epicentre, Madison, WI). The technique was based on

thepCC1FOSvector,accordingtothemanufacturer’s

instruc-tions. Escherichia coli DH5␣TM (Life Technologies, Carlsbad,

CA) transformants, carrying various recombinant fosmids,

were transferred to384 well plates.To determinean

aver-age insert size of the fosmid library, a total of 36 clones

were randomly selected and digested with the restriction

enzymeNotI.

AfractionofthelibrarywasspreadonLuria–Bertani(LB)

agar plates supplementedwithchloramphenicol (15␮g/mL)

and 0.2%carboxymethylcellulose(CMC;Sigma–Aldrich,St.

Louis,MO),whichisasubstrateforscreeningforendo-

␤-1,4-glucanaseactivity.Afterincubatedandstainedasdescribed

previously,15 fosmid clones with cellulolytic activity were

detected upon the formation of clear zones around the

colonies.Activity-basedmetagenomicscreeningyieldeda

sin-glefosmidclone(Ad125D08)thatformedoneofthe largest

clearzones.

Shotgunsequencingoftheenzyme-positivefosmidclone

Shotgunsequencingandmolecularanalyseswereperformed

to localize the cellulase gene within the enzyme-positive

fosmidcloneandtosubclonethegeneforeffective

biochem-icalcharacterization.Bacteriacarryingthecellulolyticfosmid

clone(Ad125D08)wereculturedin100mLoftheLBmedium

containing25␮g/mLofchloramphenicolat37◦Cfor20h.After

thecellswereharvested,theDNAwasextractedusinga

Qia-genPlasmidMidiKit(Qiagen,Valencia,CA)andsubjectedto

shotgunsequencingthatcomprisedtwoapproaches:

conven-tional Sangersequencing andpyrosequencing.For shotgun

sequencing with Sanger methodology, 15␮g of DNA from

thecellulolyticfosmidclonewasrandomlyfragmentedinto

sections measuring2–3kb insize. Fragmentation was

per-formedusingtheHydroShearDNAshearingdevice(Genomic

Solution,Waltham,MA)underthefollowingprocessing

con-ditions:samplevolume200␮L,speedcode11,and20shearing

cycles.Smallfragmentswereremovedandweresubsequently

repairedwithDNApolymeraseandthepolynucleotidekinase

method(BKL Kit;TaKaRa,Shiga,Japan).ThepreparedDNA

fragmentswereligatedintotheSmaIsiteofpUC19.The

liga-tion products were introduced into E. coli DH10B cells by

electroporation.Approximately192recombinantplasmidsin

theshotgunDNAlibrarywererandomlyselectedfor

sequenc-ing. Each plasmid DNA was bidirectionally sequenced as

describedpreviously.16 Thesequencingdatawasassembled

using PhredandPhrapsoftware(UniversityofWashington,

Seattle,WA).OnepyrosequencingrunwasconductedonGS

Juniorsequencingsystem(RocheDiagnostics,Oakland,CA)as

describedpreviously.17Thepyrosequencingdatawere

(3)

Insilicosequenceanalysesoftheenzyme-positivefosmid clone

All contigs from conventional shotgun sequencing with

Sangermethodologyandfromthepyrosequencingassembly

werecombinedtoconstructafullmetagenomic-insert-DNA

sequenceusingtheSeqMansoftwareinLasergeneversion7

(DNASTAR,Madison,WI).Genepredictionineachcontigwas

performedusingMetaGeneMark withdefaultparameters.18

Functional annotation was performed by searching Pfam

domains on the Pfam website (http://pfam.sanger.ac.uk).19

Amino acid sequences were deduced from the nucleotide

sequencesoftheenzyme-positive fosmidclone(Ad125D08)

andwere analyzedusingBlastPonthe NationalCenter for

BiotechnologyInformation(NCBI)databasetosearchfor

pro-teins with similarity to the amino acid sequences derived

fromtheidentifiedopenreadingframes(ORFs).Multiple

align-ments among amino acid sequences were deduced from

the ORFs, and the sequences of other proteins were

per-formedusingClustalWsoftware(http://www.ebi.ac.uk/Tools/

msa/clustalw2/). The PROSITE database was used to

pre-dictthe modulestructuresand signaturesequences ofthe

enzyme(http://prosite.expasy.org/). TheN-terminal regions

were analyzed using Signal IP software for a signal

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

and by HMMTOP 2.0 software for transmembrane regions

(http://www.enzim.hu/hmmtop/html/submit.html).

Heterologousexpressionandpurificationofthe recombinantKG35endo-ˇ-1,4-glucanase

The KG35 gene (the complete ORF of the putative

cellu-lase gene)was amplifiedfrom the enzyme-positive fosmid

clone(Ad125D08)byPCR.Theampliconwasligatedintothe

expressionvectorpET24(Novagen,Darmstadt,Germany),and

theresultingrecombinantvectorwasintroducedintoE.coli

Rosetta-gamiTM(Novagen,Darmstadt,Germany).Additional

screening for cellulolytic activity of the enzyme produced

by the transformed E. coli cells was performed using the

same CMC agar plate method as described previously.15

The cellulolytic E. coli cells were grown at 37◦C and the

transgeneexpressionwasinducedwith0.2mMisopropyl

␤-d-1-thiogalactopyranoside(IPTG)asdescribedpreviously.7After

incubation for 6h at 26◦C, the cells were harvested and

disrupted bysonication asdescribed previously.9 The

over-expressedprotein was purifiedfrom the resultingcell-free

extractsusingtheChelatingExcellose® SpinKit(Bioprogen,

Daejeon,SouthKorea).A2.0-␮Laliquotofthepurified

pro-tein was inoculated into the CMC agar plates to confirm

theenzymaticactivity.Theproteinswereseparated bySDS

polyacrylamidegelelectrophoresis(SDS-PAGE),accordingto

Laemmli(1970),20andtheproteinconcentrationswere

deter-minedbytheBradfordProteinAssayKit(Bio-Rad,Hercules,

CA).

Zymogram analysis of the recombinant KG35 endo-

␤-1,4-glucanase was performed using a 10% polyacrylamide

gel containing 0.1% CMC under denaturing conditions as

describedpreviously.21

EnzymaticcharacterizationoftherecombinantKG35 endo-ˇ-1,4-glucanase

SpecificactivitiesofthepurifiedrecombinantKG35endo-

␤-1,4-glucanasetowardvarioussubstrateswereevaluatedunder

thefollowingstandardconditions.Thesubstratesusedinthis

studywereCMC,barleyglucan,laminarin,avicel,xylanfrom

birchwood,␤-mannan,p-nitrophenyl-d-glucopyranoside,and

p-nitrophenyl-d-cellobioside. In all the assays, 10␮L of a

properly dilutedenzyme solution were added to 200␮Lof

0.1Msodium phosphate buffer (pH7),containing 1%(w/v)

of each substrate. The enzymatic reactions were allowed

to proceedat50◦C (differentreaction duration):15minfor

CMC, glucan, laminarin, and avicel; 10minfor xylanfrom

birchwood and ␤-mannan; and 20min for

p-nitrophenyl-d-glucopyranosideandp-nitrophenyl-d-cellobioside.Specific

activitywasdefinedasthenumberofactivityunitsper

mil-ligramofprotein.Oneunitofenzymaticactivitywasdefined

astheamountofenzymenecessarytorelease1␮molof

reduc-ingsugarperminute.Additionally,1unitofenzymaticactivity

producing chromogenic sugar derivatives

(p-nitrophenyl-d-glucopyranoside and p-nitrophenyl-d-cellobioside) as the

substrateswasdefinedastheamountoftheenzymerequired

torelease1␮molofp-nitrophenolperminute.Theamountsof

releasedreducingsugarandp-nitrophenolweredetermined

bymeasuringtheabsorbancesat540or400nm,respectively.

Allassayswereperformedintriplicate.

Optimal pHwas determined byassaying the enzymatic

activity of the purified recombinant, KG35 endo-

␤-1,4-glucanase,at50◦Cfor15minatpHrangingfrom4to10.To

determinetheoptimaltemperature,enzymatic activitywas

analyzedin0.1Msodiumphosphatebuffer(pH7)for15min

atatemperaturerangingfrom20to70◦C.Allassayswere

per-formedintriplicate.ThepHstabilitywasassessedafterthe

enzymewaspre-incubatedat4◦Cfor24hatpHrangingfrom

4to10.Thethermalstabilitywasassessedaftertheenzyme

waspre-incubatedfor1hin0.1Msodiumphosphatebuffer

(pH7)atvarioustemperaturesmentionedabove.Allassays

wereperformedintriplicate.

Nucleotidesequenceaccessionnumber

Thenucleotidesequenceoftheenzyme-positivefosmidclone

(Ad125D08), including the complete ORF ofthe KG35 gene

(anovelGH5cellulasegene),wasdepositedintheGenBank

databaseundertheaccessionnumberKJ631389.

Results

Constructionandfunctionalscreeningofthemetagenomic fosmidlibrary

Ametagenomicfosmidlibrarycontaining115,200cloneswas

constructedfromtherumenmicroorganismsofblackgoats.

NotI restriction enzymeanalysesrevealed that the average

insertsizewastypically31kb(datanotshown).Atotalof155

independentfosmidclones,displayingcarboxymethyl

cellu-lase(CMCase)activity,wereidentified.Amongthem,asingle

(4)

ontheCMC-containingscreeningplatesandwasselectedfor

furtheranalysis.

ConventionalshotgunsequencingwiththeSangermethod

wasperformedunder2.7-foldcoverage,and∼300kbof

pyrose-quencingreadswereproduced.Consequently,onecontigof

42.453kbwasobtainedwithoutthepCC1FOSvectorsequence.

Thirty-fourORFs were predicted in the contig sequence of

fosmidcloneAd125D08,and anORF containingacellulase

domain(KG35gene)wasidentifiedandlocalizedwithinthe

clone.

InsilicosequenceanalysisoftheputativeKG35 endo-ˇ-1,4-glucanase

SequenceanalysisoftheputativeKG35generevealeda

sin-gleORFof963bpencodingapolypeptideof320aminoacid

residues,withanestimatedmolecularmassof35.1kDaand

pIof5.SignalIPandHMMTOP2.0softwareprogramscouldnot

detectanyknownsignalpeptidesequenceortransmembrane

regionintheKG35protein.Themodulestructuresearchinthe

PROSITEdatabasepredictedthattheKG35proteincontainsa

GH5cellulasecatalyticdomainwithaconserved(aminoacid)

signaturesequenceVLYEICNEPN,anditssecondGluresidue

wasassumedtoplayacatalyticrole(Fig.1).22,23

ABlastPsearch,comparingtheaminoacidsequenceofthe

KG35 proteinwiththose ofother proteinsregisteredinthe

GenBankdatabase,revealedthatthisproteinhastheclosest

(58%)sequenceidentitytoahypotheticalproteinfrom

Eubac-terium cellulosolvens (WP 051527028.1) and shares even less

sequence identitywithother GH5family proteinhomologs

from various microbial species (Table 1). The amino acid

sequencesofthesehomologousproteinspossess

character-istic featuresconservedamongthe GH5family ofenzymes

(Fig.1).

PurificationandSDS-PAGEanalysesoftherecombinant KG35endo-ˇ-1,4-glucanase

The putative endo-␤-1,4-glucanase gene(KG35) was cloned

and overexpressed inE. coliand theresulting recombinant

enzymewaspurified,asdescribedinthematerialsand

meth-odssection.SDS-PAGEanalysisofthepurifiedrecombinant

KG35endo-␤-1,4-glucanaseshowedasinglebandonthegel,

withtheapparentmolecularmassofapproximately 35kDa

(Fig.2).Zymogramanalysisrevealedasingleband

correspond-ingtoanactiveendo-␤-1,4-glucanaseofthesamemolecular

massastheband(Fig.2).

Fig.1–MultiplealignmentoftheKG35endo-␤-1,4-glucanaseGH5domainwithotherhomologousGH5proteindomains. ThededucedaminoacidsequenceofaGH5familydomaininKG35endo-␤-1,4-glucanasewasalignedwiththoseof selectedhomologousenzymesfromthefollowingmicroorganisms:Eubacteriumcellulosolvens(WP051527028.1);

Agathobacterrectalis(WP055222932.1);Roseburiainulinivorans(WP055169535.1);A.rectalis(WP012741522.1);Ruminococcus torques(WP055163516.1);EubacteriumrectaleCAG:36(CDC71888.1);R.inulinivorans(WP055040325.1);andLachnospiraceae bacteriumC6A11(WP051646390.1).SignaturesequencesofGH5domainsareshadedingray.TheGluresiduesunderthe starsymbolsindicatethecatalyticsites.

(5)

Table1–AminoacidsequenceidentitybetweenthenovelKG35endo-␤-1,4-glucanaseandother(Homologous)proteins.

Proteinname (Organismname)

GeneBankaccessionnumber Querycoverage (%)a Identity (%)a Hypotheticalprotein (Eubacteriumcellulosolvens) WP 051527028.1 98 58

Glycosylhydrolasefamily5protein

(Agathobacterrectalis)

WP055222932.1 98 52

Glycosylhydrolasefamily5protein

(Roseburiainulinivorans)

WP055169535.1 98 51

Endo-1,4-␤-glucanase (A.rectalis)

WP012741522.1 98 51

Glycosylhydrolasefamily5protein

(Ruminococcustorques)

WP055163516.1 98 51

Endoglucanase

(EubacteriumrectaleCAG:36)

CDC71888.1 98 51

Glycosylhydrolasefamily5protein

(R.inulinivorans)

WP055040325.1 98 50

Hypotheticalprotein

(LachnospiraceaebacteriumC6A11)

WP051646390.1 98 50

a ThenovelKG35endo-␤-1,4-glucanasegenewasusedforBlastPsearchesusingthe98%querycoveragecutoffvalueand50%identitycutoff

value.

Table2–SubstratespecificityofthenovelKG35endo-␤-1,4-glucanasetowardvarioussubstrates.

Substrate Mainlinkagetypes Specificactivity(U/mg) Carboxylmethylcellulose(CMC) ␤-1,4-Glucan 8.40±0.03

Barleyglucan ␤-1,3/1,4-Glucan 5.40±0.19

P-nitrophenyl-d-cellobioside ␤-1,4-Glucan 0.51±0.01

Laminarin ␤-1,3/1,6-Glucan 0.48±0.04

Avicel ␤-1,4-Glucan 0.12±0.02

Xylanfrombirchwood ␤-1,4-Xylan 0.00±0.00

␤-Mannan ␤-1,4-Mannan 0.00±0.00 P-nitrophenyl-d-glucopyranoside ␤-1,4-Glucan 0.00±0.00 M 1 2 3 4 75 kDa 63 kDa 48 kDa 35 kDa 28 kDa

Fig.2–SDS-PAGEanalysesofrecombinantKG35

endo-␤-1,4-glucanaseproducedinE.coliRosetta-gamiTM.

LaneM:proteinmolecularweightmarkers;lane1:total

cell-freeextractsbeforeinduction;lane2:thesoluble

fractionaftertheinduction;lane3:purifiedKG35

endo-␤-1,4-glucanase;lane4:zymogramanalysisofKG35

endo-␤-1,4-glucanase.

CharacterizationofthepurifiedrecombinantKG35 endo-ˇ-1,4-glucanase

Substrate specificity toward various substrates was deter-mined (Table 2). The endo-␤-1,4-glucanase showed strong

specific activities toward CMC (8.40U/mg) and barley

glu-can (5.40U/mg). In contrast, this enzyme showed only

trace enzymatic activity toward laminarin (0.48U/mg),

p-nitrophenyl-d-cellobioside(0.51U/mg),andavicel(0.12U/mg).

In addition, the endo-␤-1,4-glucanase could not hydrolyze

p-nitrophenyl-d-glucopyranoside,xylanfrombirchwood,or ␤-mannan.

The optimal pH and temperature of the recombinant

enzyme were determined under various pH and

tempera-tureconditions(Fig.3).TheoptimalpHandtemperatureof

therecombinantKG35endo-␤-1,4-glucanasetowardCMCwas

approximately6–7and30–50◦C,respectively.

In addition, the enzymatic stability was tested under

variouspHandtemperatureconditions(Fig.4).Theendo-

␤-1,4-glucanase retainedmorethan 70%ofactivity after24h

of incubation at pH 5–7. The same enzyme showed

ther-mostability after a 1-h incubation at 30–50◦C; however, it

declinedinactivitybygreaterthan 60%aftera1-h

incuba-tionabove50◦C.Ourempiricaldatacollectivelyindicatesthat

therecombinantenzymeretaineditspHandtemperature

sta-bilityapproximatelywithinitsoptimalpHandtemperature

ranges,respectively.

Discussion

Ithasbeenreportedthatthe GH5family ofcellulases

(6)

120 100 80 60 40 20 0

A

B

Relativ e activity (%) 3 4 5 6 7 8 9 10 11 10 20 30 40 50 60 70 80 pH 120 100 80 60 40 20 0 Relativ e activity (%) Temperature (ºC)

Fig.3–EffectsofpHandtemperatureontheactivityofrecombinantKG35endo-␤-1,4-glucanase.(A)Theoptimalenzyme activitywasmeasuredatapHrangeof4–10.Thebufferswere0.1Msodiumacetate(pH4–6,䊉),0.1Msodiumphosphate (pH6–8,),0.1MTris–HCl(pH8–9,),and0.1Mglycine–NaOH(pH9–10,).(B)Theoptimalenzymeactivitywasmeasured atatemperaturerangeof20–70◦C.Theerrorbarsrepresentthestandarddeviationoftriplicatemeasurements.

120 100 80 60 40 20 0

A

B

Relativ e activity (%) 3 4 5 6 7 8 9 10 11 10 20 30 40 50 60 70 80 pH 120 100 80 60 40 20 0 Relativ e activity (%) Temperature (ºC)

Fig.4–EffectsofpHandtemperatureonthestabilityoftherecombinantKG35endo-␤-1,4-glucanase.(A)ThepHstabilityas determinedbymeasuringtheresidualactivityaftertheenzymewaspreincubatedat4◦Cfor24hattheindicatedpHlevels from4to10.Thebufferswere0.1Msodiumacetate(pH4–6,䊉),0.1Msodiumphosphate(pH6–8,),0.1MTris–HCl(pH8–9,

),and0.1Mglycine–NaOH(pH9–10,).(B)Thetemperaturestabilityasdeterminedbymeasuringtheresidualactivityafter theenzymewaspre-incubatedatpH7for1hoverthetemperaturerangeof20–70◦C.Theerrorbarsrepresentthestandard deviationoftriplicatemeasurements.

[LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-

{SENR}-N-E-[PV]-[RHDNSTLIVFY].24Theconservedsignaturesequenceofthe

KG35proteincouldbelocatedintheabove-mentionedGH5

familysignaturesequencepattern.Thisresultindicatedthat

theKG35proteinexhibitedastrongrelationtotheGH5family

of cellulases. Furthermore, the KG35 endo-␤-1,4-glucanase

protein exhibited the closest (58%) amino acid sequence

similaritytoitsGH5homologousenzymes,implyingthatthe

KG35endo-␤-1,4-glucanaseproteinhasneverbeenidentified

orcharacterized,thusisanovelaminoacidsequence.

TheCMCandbarleyglucan,whichareprimarilybasedon

␤-1,4-glycosidicbondsandhaveamorphousstructures,were

foundtobethemostfavorablesubstratesfortherecombinant

KG35endo-␤-1,4-glucanase.Amorphouslaminarin,

contain-ing␤-1,3/1,6-glycosidicbonds,andhemicelluloses,including

xylan from birchwood and ␤-mannan, were not specific

substrates of the recombinant KG35 endo-␤-1,4-glucanase.

Althoughavicelmostlycontains␤-1,4-glycosidicbonds,itwas

notefficientlyhydrolyzedbythisenzyme.Avicelcontains

␤-1,4-glycosidicbondsakintoothersolublecellulosicsubstrates,

anditscrystallinestructurecanundergoefficienthydrolysis

whenacellulose-bindingdomainisinvolvedinthe

hydrol-ysisreaction.25,26 Nevertheless,sequenceanalysiscouldnot

detectanycellulose-bindingdomainintheKG35endo-

␤-1,4-glucanase;thereforetheabsenceofthisdomainmayexplain

theweakenzymaticactivitytowardavicel.Twosynthetic

sub-strates usedinthis study,p-nitrophenyl-d-cellobioside and

p-nitrophenyl-d-glucopyranoside,aresyntheticsubstratesfor

exoglucanaseand␤-glucosidase,respectively.27,28 Therefore,

the recombinant KG35 endo-␤-1,4-glucanase exerted slight

exoglucanase activityandno␤-glucosidase activity,

respec-tively. Alloftheseresultspertainingtosubstratespecificity

indicate thatKG35cellulase ismostlikelytohydrolyzethe

␤-1,4-glycosidicbondsoftheamorphousregionsofcellulose

andconsequentlyfunctionasanendo-␤-1,4-glucanase.

TherecombinantKG35cellulasewasmaximallyactiveand

enzymaticallystableatapHrangeof6–7andatatemperature

rangeof30–50◦C.Theoptimumandstabilitycharacteristics

ofthe recombinant enzymewere similarto those ofother

(7)

ruminants.Othercellulasesfromrumenalmicroorganismsof

thecowwerereportedtohavetheoptimalpHrangeof5–7

andanoptimaltemperatureofapproximately50◦C.9,29,30The

cellulasefromrumen-residentbacteriawasreportedtoretain

70%ofitsactivityinthepHrangefrom5to7andina

temper-aturerangefrom30to50◦C.30

TheruminalpHofsmallruminantsranges from 5to7,

undernormal dietaryconditions, and the ruminal

temper-aturecanvary from38to41◦C.31 Thecellulolyticreactions

ofruminal-bacteria cellulase havebeen reportedto be

rel-ativelymoreresistantatthepHof6.5–7inculture,butthe

reactionswereweakenedastheculturepHdecreased.32KG35

endo-␤-1,4-glucanase may be well-adapted to the ruminal

environmentsofblackgoatsbecausethepHandtemperature

conditionsunder whichthe recombinant KG35 endo-

␤-1,4-glucanaseshowedrelativelyhigheractivityandstabilitywere

inagreementwiththeruminalpHandtemperatureconditions

thatoccursnaturallyinruminants,includinggoats.Theabove

observation can explainwhy the recombinant KG35

endo-␤-1,4-glucanasefunctionsbetteratmildly acidic/neutralpH

andamoderatetemperaturethanatanacidic/alkalinepHor

stronglyunphysiologicaltemperature.

Cellulase can effectively convert cellulose, an

anti-nutritional component, in animal feedstocks into its

hydrolysates, nutritive ingredients, yielding improved

per-formance of animals.5 Therefore, applications of different

cellulasesfrom varioussourceshaveattractedconsiderable

attentions inthe feed industry.KG35 endo-␤-1,4-glucanase

maybeexploitedasafeedadditivetoimprovefeedutilization

ofruminants, becausethe enzymemay bewell-adaptedto

the ruminal environments. Moreover, the enzyme can be

potentiallyusedtomakecellulosicethanol,second

genera-tionbiofuel,fromcellulosicfeedstockssuchasgrass,wood,

andcropresidues.

Thisstudypresents novelresultspertaining tothe

con-structionand screeningofametagenomic libraryfrom the

rumenalmicroorganismsofblackgoatsforidentificationand

characterizationofanovelcellulolyticenzyme.Atotalof155

cellulolytic fosmid clones were selected from the

metage-nomiclibrary.Amongthem,theKG35genewassuccessfully

isolated as a clone with the strongest cellulose-degrading

activity.TheKG35genewasfoundtoencodeanovelmember

oftheGH5familyofcellulases,andthisnovelgenewas

effi-cientlytranslatedandoverexpressedinE.coliRosetta-gamiTM.

Substratespecificityanalysisrevealedthattherecombinant

KG35 cellulase functions as an endo-␤-1,4-glucanase. The

recombinantKG35endo-␤-1,4-glucanaseoptimallyfunctions

inthepHrangeof6–7andtemperaturerangeof30–50◦C.The

endo-␤-1,4-glucanaseretainedenzymaticstabilityinthepH

rangeof5–7andthetemperaturerangeof30–50◦C.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thisworkwascarriedoutwiththesupportof“Cooperative

ResearchPrograms forAgriculture Scienceand Technology

Development(ProjectNos.PJ006649,PJ008701andPJ011163)”,

RuralDevelopmentAdministration,RepublicofKorea.J.-S.Lee

(forinsilicoexperimentdesign)andK.-S.Kim(forother

exper-imentdesign)areco-correspondingauthorsofthiswork.

r

e

f

e

r

e

n

c

e

s

1.BhatMK,BhatS.Cellulosedegradingenzymesandtheir

potentialindustrialapplications.BiotechnolAdv.

1997;15:583–620.

2.LyndLR,WeimerPJ,vanZylWH,PretoriusIS.Microbial

celluloseutilization:fundamentalsandbiotechnology.

MicrobiolMolBiolRev.2002;66(3):506–577.

3.BhatMK.Cellulasesandrelatedenzymesinbiotechnology.

BiotechnolAdv.2000;18(5):355–383.

4.SweeneyMD,XuF.Biomassconvertingenzymesas

industrialbiocatalystsforfuelsandchemicals:recent

developments.Catalysts.2012;2:244–263.

5.KuhadRC,GuptaR,SinghA.Microbialcellulasesandtheir

industrialapplications.EnzymeRes.2011;2011:1–10.

6.KrauseDO,DenmanSE,MackieRI,etal.Opportunitiesto

improvefiberdegradationintherumen:microbiology,

ecology,andgenomics.FEMSMicrobiolRev.

2003;27(5):663–693.

7.FengY,DuanCJ,PangH,etal.Cloningandidentificationof

novelcellulasegenesfromunculturedmicroorganismsin

rabbitcecumandcharacterizationoftheexpressed

cellulases.ApplMicrobiolBiotechnol.2007;75(2):319–328.

8.DuanCJ,XianL,ZhaoGC,etal.Isolationandpartial

characterizationofnovelgenesencodingacidiccellulases

frommetagenomesofbuffalorumens.JApplMicrobiol.

2009;107(1):245–256.

9.NguyenNH,MarusetL,UengwetwanitT,etal.Identification

andcharacterizationofacellulase-encodinggenefromthe

buffalorumenmetagenomiclibrary.BiosciBiotechnolBiochem.

2012;76(6):1075–1084.

10.AmannRI,LudwigW,SchleiferKH.Phylogenetic

identificationandinsitudetectionofindividualmicrobial

cellswithoutcultivation.MicrobiolRev.1995;59(1):143–169.

11.MireteS,MorganteV,González-PastorJE.Functional

metagenomicsofextremeenvironments.CurrOpin

Biotechnol.2016;38:143–149.

12.LeeID,LeeHS.StudyonthefoodhabitsofKoreannative

goat(Caprahircus)fedwithvariousroughagesources.JKor

SocGrasslForageSci.2008;38:445–452.

13.HofmannRR,StewartDRM.Grazerorbrowser:a

classificationbasedonthestomachstructureandfeeding

habitsofEastAfricanruminants.Mammalia.1972;36:226–240.

14.ZhouJ,BrunsMA,TiedjeJM.DNArecoveryfromsoilsof

diversecomposition.ApplEnvironMicrobiol.

1996;62(2):316–322.

15.GengA,ZouG,YanX,etal.Expressionandcharacterization

ofanovelmetagenome-derivedcellulaseExo2bandits

applicationtoimprovecellulaseactivityinTrichodermareesei.

ApplMicrobiolBiotechnol.2012;96(4):951–962.

16.LeeKT,ByunMJ,KangKS,etal.Neuronalgenesfor

subcutaneousfatthicknessinhumanandpigareidentified

bylocalgenomicsequencingandcombinedSNPassociation

study.PLoSONE.2011;6(2):e16356.

17.ChoiJW,LiaoX,StothardP,etal.Whole-genomeanalysesof

KoreannativeandHolsteincattlebreedsbymassively

parallelsequencing.PLOSONE.2014;9(7):e101127.

18.ZhuW,LomsadzeA,BorodovskyM.Abinitiogene

identificationinmetagenomicsequences.NucleicAcidsRes.

(8)

19.PuntaM,CoggillPC,EberhardtRY,etal.ThePfamprotein

familiesdatabase.NucleicAcidsRes.2012;40:D290–D301.

20.LaemmliUK.Cleavageofstructuralproteinsduringthe

assemblyoftheheadofbacteriophageT4.Nature.

1970;227(5259):680–685.

21.LeeCM,LeeYS,SeoSH,etal.Screeningandcharacterization

ofanovelcellulasegenefromthegutmicrofloraofHermetia

illucensusingmetagenomiclibrary.JMicrobiolBiotechnol.

2014;24(9):1196–1206.

22.HenrissatB,CallebautI,FabregaS,LehnP,MornonJP,Davies

G.Conservedcatalyticmachineryandthepredictionofa

commonfoldforseveralfamiliesofglycosylhydrolases.Proc

NatlAcadSciUSA.1995;92(15):7090–7094.

23.WangQ,TullD,MeinkeA,etal.Glu280isthenucleophilein

theactivesiteofClostridiumthermocellumCelC,afamilyA

endo-beta-1,4-glucanase.JBiolChem.

1993;268(19):14096–14102.

24.CheemaTA,JirajaroenratK,SirinarumitrT,RakshitSK.

Isolationofageneencodingacellulolyticenzymefrom

swampbuffalorumenmetagenomesanditscloningand

expressioninEscherichiacoli.AnimBiotechnol.

2012;23(4):261–277.

25.LeCostaouëcT,PakarinenA,VárnaiA,PuranenT,ViikariL.

Theroleofcarbohydratebindingmodule(CBM)athigh

substrateconsistency:comparisonofTrichodermareeseiand

ThermoascusaurantiacusCel7A(CBHI)andCel5A(EGII).

BioresourTechnol.2013;143:196–203.

26.ZhaoL,PangQ,XieJ,PeiJ,WangF,FanS.Enzymatic

propertiesofThermoanaerobacteriumthermosaccharolyticum

␤-glucosidasefusedtoClostridiumcellulovoranscellulose

bindingdomainanditsapplicationinhydrolysisof

microcrystallinecellulose.BMCBiotechnol.2013;13:101.

27.MallermanJ,PapinuttiL,LevinL.Characterizationof

␤-glucosidaseproducedbythewhiterotfungusFlammulina

velutipes.JMicrobiolBiotechnol.2015;25(1):57–65.

28.JingL,ZhaoS,XueJ,etal.Isolationandcharacterizationofa

novelPenicilliumoxalicumstrainZ1-3withenhanced

cellobiohydrolaseproductionusingcellulase-hydrolyzed

sugarcanebagasseascarbonsource.IndCropsProd.

2015;77:666–675.

29.ZhaoS,WangJ,BuD,etal.Novelglycosidehydrolases

identifiedbyscreeningaChineseHolsteindairycow

rumen-derivedmetagenomelibrary.ApplEnvironMicrobiol.

2010;76(19):6701–6705.

30.GongX,GruningerRJ,QiM,etal.Cloningandidentification

ofnovelhydrolasegenesfromadairycowrumen

metagenomiclibraryandcharacterizationofacellulase

gene.BMCResNotes.2012;5:566–576.

31.MackieRI,McSweeneyCS,AminovRI.Rumen.In:

EncyclopediaofLifeSciences.Chichester,England:JohnWiley

andSons;2013.

32.RussellJB,DombrowskiDB.EffectofpHontheefficiencyof

growthbypureculturesofrumenbacteriaincontinuous

Referências

Documentos relacionados

Resumo das análises de variância para a variável altura da planta AT, diâmetro caulinar DC, número de folhas NF, diâmetro da cabeça DCA e massa fresca MF em função das fontes

social assistance. The protection of jobs within some enterprises, cooperatives, forms of economical associations, constitute an efficient social policy, totally different from

The probability of attending school four our group of interest in this region increased by 6.5 percentage points after the expansion of the Bolsa Família program in 2007 and

Portanto, além de discutir e enlaçar as categorias de gênero, beleza e relações de trabalho, será realizada ainda uma pesquisa de campo a fim de evidenciar como

Na hepatite B, as enzimas hepáticas têm valores menores tanto para quem toma quanto para os que não tomam café comparados ao vírus C, porém os dados foram estatisticamente

The characters analysed included effect of pH (4 - 10) and temperature (30 - 80 o C) on enzyme activity and stability, effect of NaCl (0 - 20%) on enzyme activity, effect of

Effects of temperature (A) and pH (B) on the activity of lactose hydrolyzed enzyme in cell free extracts of strain KNOUC808. * Effect of temperature was tested in Na-phosphate

In the present research project, lactobacilli strains producing bacteriocin effective against cephalosporin resistant Escherichia coli were isolated and characterized