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,SouthKoreabAnimalGenomicsandBioinformaticsDivision,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
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 (15g/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
containing25g/mLofchloramphenicolat37◦Cfor20h.After
thecellswereharvested,theDNAwasextractedusinga
Qia-genPlasmidMidiKit(Qiagen,Valencia,CA)andsubjectedto
shotgunsequencingthatcomprisedtwoapproaches:
conven-tional Sangersequencing andpyrosequencing.For shotgun
sequencing with Sanger methodology, 15g of DNA from
thecellulolyticfosmidclonewasrandomlyfragmentedinto
sections measuring2–3kb insize. Fragmentation was
per-formedusingtheHydroShearDNAshearingdevice(Genomic
Solution,Waltham,MA)underthefollowingprocessing
con-ditions:samplevolume200L,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
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, 10L of a
properly dilutedenzyme solution were added to 200Lof
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
astheamountofenzymenecessarytorelease1molof
reduc-ingsugarperminute.Additionally,1unitofenzymaticactivity
producing chromogenic sugar derivatives
(p-nitrophenyl-d-glucopyranoside and p-nitrophenyl-d-cellobioside) as the
substrateswasdefinedastheamountoftheenzymerequired
torelease1molofp-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
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.
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
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
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.
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