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RESEARCHOpenAccessThermoanaerobacteriumthermosaccharolyticumβ-glucosidase:aglucose-tolerantenzymewithhighspecificactivityforcellobioseJianjunPei1,2,QianPang1,2,LinguoZhao1,2*,SongFan1,2andHaoShi1,2AbstractBackground:β-Glucosidaseisanimportantcomponentofthecellulaseenzymesystem.
Itdoesnotonlyparticipateincellulosedegradation,italsoplaysanimportantroleinhydrolyzingcellulosetofermentableglucosebyrelievingtheinhibitionofexoglucanaseandendoglucanasefromcellobiose.
Therefore,theglucose-tolerantβ-glucosidasewithhighspecificactivityforcellobiosemightbeapotentcandidateforindustrialapplications.
Results:Theβ-glucosidasegenebglthatencodesa443-amino-acidproteinwasclonedandover-expressedfromThermoanaerobacteriumthermosaccharolyticumDSM571inEscherichiacoli.
Thephylogenetictreesofβ-glucosidaseswereconstructedusingNeighbor-Joining(NJ)andMaximum-Parsimony(MP)methods.
ThephylogenyandaminoacidanalysisindicatedthattheBGLwasanovelβ-glucosidase.
ByreplacingtherarecodonsfortheN-terminalaminoacidsofthetargetprotein,theexpressionlevelofbglwasincreasedfrom6.
6to11.
2U/mginLBmedium.
RecombinantBGLwaspurifiedbyheattreatmentfollowedbyNi-NTAaffinity.
TheoptimalactivitywasatpH6.
4and70°C.
ThepurifiedenzymewasstableoverpHrangeof5.
2–7.
6andhada1hhalflifeat68°C.
TheactivityofBGLwassignificantlyenhancedbyFe2+andMn2+.
TheVmaxof64U/mgand120U/mgwerefoundforp-nitrophenyl-β-D-glucopyranoside(Kmvalueof0.
62mM)andcellobiose(Kmvalueof7.
9mM),respectively.
Itdisplayedhightolerancetoglucoseandcellobiose.
TheKcatforcellobiosewas67.
7s-1at60°CandpH6.
4,whentheconcentrationofcellobiosewas290mM.
Itwasactivatedbyglucoseatconcentrationslowerthat200mM.
Withglucosefurtherincreasing,theenzymeactivityofBGLwasgraduallyinhibited,butremained50%oftheoriginalvalueinevenashighas600mMglucose.
Conclusions:Thearticleprovidesausefulnovelβ-glucosidasewhichdisplayedfavorableproperties:highglucoseandcellobiosetolerance,independenceofmetalions,andhighhydrolysisactivityoncellobiose.
Keywords:β-glucosidase,Glucosetolerance,Thermoanaerobacteriumthermosaccharolyticum,Over-expression,PhylogenyIntroductionCellulosicbiomassisthemostabundantrenewableresourceonearth,whosenaturaldegradationrepresentsanimportantpartofthecarboncyclewithinthebio-sphere[1].
β-Glucosidase(EC3.
2.
1.
21)isaglucosidaseenzymethatactsuponβ1–4bondslinkingtwoglucoseorglucose-substitutedmolecules.
Itisanimportantcomponentofthecellulaseenzymesystem.
Thelimitingstepintheenzymaticsaccharificationofcellulosicmater-ialistheconversionofshort-chainoligosaccharidesandcellobiose,whichwasresultedfromthesynergisticactionofendogucanases(EC3.
2.
1.
4)andcellobiohydrolases(EC3.
2.
1.
91),toglucose,areactioncatalyzedbyβ-glucosidases[2].
Itiswellestablishedthatcellobioseinhibitstheactiv-itiesofmostcellobiohydrolasesandendoglucanses[3].
β-glucosidasesreducecellobioseinhibitionbyhydrolyzingthisdisaccharidetoglucose,thusallowingthecellulolyticenzymestofunctionmoreefficiently[4,5].
Furthermore,β-glucosidaseisusedasaflavorenzymetoenhancetheflavorofwine,teaandfruitjuice[6,7].
Infruitsandotherplanttissuesmanysecondarymetabolites,includingflavor*Correspondence:lg.
zhao@163.
comEqualcontributors1CollegeofChemicalEngineering,NanjingForestryUniversity,Nanjing,210037,China2JiangsukeyLabofBiomassBasedGreenFuelsandChemicals,Nanjing,China2012Peietal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Peietal.
BiotechnologyforBiofuels2012,5:31http://www.
biotechnologyforbiofuels.
com/content/5/1/31compounds,areaccumulatedintheirglucosylatedform[8,9].
Becauseβ-glucosidesconstitutethemajorityoftheknownglycoconjugatedflavorcompounds,β-glucosidasesplayanimportantroleinflavorliberationfromthesepre-cursors.
Therefore,producinghigh-activityandglucose-tolerantβ-glucosidasehasbecomeimportant.
Recently,thesearchforβ-glucosidasesinsensitivetoglucosehasincreasedsignificantly,fortheseenzymeswouldimprovetheprocessofsaccharificationofligno-cellulosicmaterials.
Afewmicrobialβ-glucosidaseshavebeenreportedtotolerateglucose[10-14].
Forexample,β-glucosidasesfromAspergillustubingensisCBS643.
92,A.
oryzae,A.
nigerCCRC31494,A.
foetidus,andmarinemicrobialmetagenomedisplayedhighinhibitionconstantbyglucose(Ki)of600mM,1390mM,543mM,520mM,and1000mM,respectively.
Buttheseβ-glucosidaseshaveconsiderablylowerspecificactivityforcellobiosethanforp-nitrophenyl-β-D-glucopyranoside.
Therefore,over-expressionofthermostableβ-glucosidasewithhighglucosetoleranceandspecificactivityforcellobioseabilitieswillhelpshedlightondegradationofcellu-losicbiomass.
Thermostableenzymeshaveseveralgenericadvantages,allowingadecreasedamountofenzymeneededbecauseofhigherspecificactivityandelongatedhydrolysistimeduetohigherstability.
Inaddition,thermostableenzymesaregenerallymoretolerantandallowmoreflexibilityinprocessconfigurations[15,16].
Althoughsomeglucose-tolerantβ-glucosidasesfromfungiandbacteriahavebeenreported[10-14],theglucose-tolerantβ-glucosidasesgeneshavenotbeenexpressedandcharacterizedfromthermo-philicbacteria.
BacteriumThermoanaerobacteriumther-mosaccharolyticumisastrictanaerobethatgrowsonwiderangeofhexoseandpentoseattemperaturefrom37°Cto75°C,whichhaveattractedconsiderableintereststohydro-genproductionandthermostableenzymeproduction[17].
T.
thermosaccharolyticumDSM571couldutilizecello-biose,butthegeneforβ-glucosidase,thekeyenzymeindegradationcellobiose,wasnotreportedintheGenbank(NC_014410.
1).
BecausetheoptimalgrowthtemperatureforT.
thermosaccharolyticumDSM571wasat60°C,thethermostableβ-glucosidasecouldhaveaconsiderablepo-tentialforindustrialapplications.
OwingtotheinherentdifficultyofcultivationofT.
thermosaccharolyticumDSM571,itisdifficulttoobtainasufficientamountofcellsforlarge-scaleenzymeproduction.
Fortheproductionoftherecombinantprotein,geneticengineeringisthefirstchoicebecauseitiseasy,fast,andcheap.
Inthispaper,wereportthephylogenesisanalysis,cloning,over-expression,anddetailedbiochemicalchar-acterizationoftheβ-glucosidasefromT.
thermosaccharo-lyticumDSM571.
Thefavorablepropertiesmaketheβ-glucosidaseagoodcandidateforutilizationinbiotech-nologicalapplications.
ResultsCloningandsequenceanalysisofbglByanalysisofthegenomesequenceofT.
thermosaccharo-lyticumDSM571,aprotein(Tthe_1813),definedasβ-galactosidaseinGenbank,consistsofa1,329-bpfragmentencoding443aminoacids,whichbelongedtofamily1oftheglycosidehydrolases.
Itsharesthehighestsequencesimilarityof66%withtheβ-glucosidsesfromThermoa-naerobactermathranii(GenbankNo.
YP_003676178.
1)andThermoanaerobacterpseudethanolicusATCC33223(GenbankNo.
YP_001665894.
1),whichwererevealedbywhole-genomesequencingbuthasnotbeenbiochemicallycharacterized.
AlignmentoftheBGLclusterwithseveralrepresentativemembersofGH1indicatedthattheysharesimilarblocks.
Thecatalyticprotondonor,Glu135andGlu351inBGLarewellconservedamongallGH1pro-teins(Figure1).
ThesequencearoundGlu351inBGLis[LYT-NGAA],whichisconsistentwiththeconsensuspat-ternofPS00572.
Theresultsindicatedthattheprotein(Tthe_1813)couldbeanovelβ-glucoside.
ThentheDNAfragmentofaprotein(Tthe_1813)genewasamplifiedfromgenomicDNAofT.
thermosaccharolyticumDSM571,andligatedtopET-20batNdeIandXhoIsitestogenerateplasmidpET-20-BGL.
Over-expressionofBGLInordertoincreasetheexpressionlevelofBGLinE.
coli,site-directedmutagenesisweredesignedandperformedtooptimizecondonsofBGLforE.
coliexpressionsystem.
pET-20-BGLIIwasobtainedfrompET-20-BGLinwhichtherarecondonsfortheN-terminalaminoacidresidueswerereplacedbyoptimalcodonsinE.
coliwithoutandchangeofaminoacidsequence(Figure2),sopET-20-BGLIIencodesthesameβ-glucosidaseasthatencodedbythewild-typegene.
Theβ-glucosidaseactivityexpres-sionfrompET-20-BGLIIwas7.
5U/mL(11.
2U/mgtotalofcellprotein)andwasestimatedtobeabout30%ofthetotalprotein,whichwasabout1.
7timeshigherthantheexpressedfrompET-20-BGL(Figure3,lane2and3).
PurificationandCharacterizationofrecombinantBGLTheproteininthecell-freeextractwaspurifiedtogelelectrohomogeneityafteraheattreatmentandaNi-NTAaffinity.
ThefinalpreparationgaveasinglebandonSDS-PAGEgelandthemolecularmassoftheenzymewasestimatedtobe52kDa(Figure3,lane4).
ThebiochemicalpropertiesofBGLwereinvestigatedbyusingthepurifiedrecombinantBGL.
TheoptimalpHoftheBGLwasdeterminedtobe6.
4(Figure4a),whiletheβ-glucosidaseactivitywashigherthan50%ofthemaximumactivityatthepHrangefrom5.
6to7.
2.
Theenzymewasstableforabout1hatpH5.
6to8.
0at60°Cintheabsenceofthesubstrate(Figure4c).
TheoptimaltemperatureofPeietal.
BiotechnologyforBiofuels2012,5:31Page2of10http://www.
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com/content/5/1/31theBGLwas70°C,whichtheβ-glucosidaseactivitywashigherthan40%ofthemaximumactivityatthetemperaturerangefrom45to75°C(Figure4b).
Ther-mostabilityassaysindicatedthatitsresidualactivitywasmorethan80%afterbeingincubatedat60°Cfor2h(pH6.
4,Figure4d).
Theeffectsofmetalionsandsomechemicalsontheenzymeactivitywereshownin(Table1).
Invariousassays,theenzymeactivitywassignificantlyenhancedbyFe2+,orMn2+,andcompletelyinactivatedbyZn2+,Cu2+,Ag2+,orHg2+.
TheeffectsofMg2+,Ca2+,K+,Li2,orEDTA(10mM)ontheenzymeactivitywerenotsosignificant.
EffectofglucoseonBGLactivityandsubstratespecificityTheenzymewasabletohydrolyzep-nitrophenyl-β-D-glucopyranoside,cellobiose,andp-nitrophenyl-β-D-galactopyranoside,whilenoactivitywasdetecteduponp-nitrophenyl-α-L-arabinofuranoside,p-nitrophenyl-β-D-xylopyranoside,maltose,CMC,andsucrose.
p-nitrophenyl-β-D-Galactopyranosidewashydrolyzedat40%ofthatofp-nitrophenyl-β-D-glucopyranoside.
ThedependenceoftheFigure2Thecodonsfortheaminoacidbetweenthe1stand19thwhichweresubjectedtosite-directedmutagenesis.
OriginalsequenceoftheBGL(opensquare);optimalsequenceoftheBGL(filledsquare).
Figure1MultiallignmentofBGLwithsomeGH1familymembers.
SequencealignmentwasperformedbyusingClustalX2.
0.
Theactivesitesareindicatedas*onthetopofthealignment.
T.
t:T.
thermosaccharolyticumDSM571(YP_003852393.
1),T.
a:Trichodermaatroviride(EHK41167.
1),T.
m:Thermotogamaritima(Q08638.
1),A.
o:Aspergillusoryzae(BAE57671.
1),A.
f:Aspergillusfumigatus(XP_752840.
1).
Peietal.
BiotechnologyforBiofuels2012,5:31Page3of10http://www.
biotechnologyforbiofuels.
com/content/5/1/31rateoftheenzymaticreactiononthesubstratesconcentra-tionfollowedMichaelis-Mentenkinetics,withKmandVmaxvaluesof0.
62mMand64U/mgforp-nitrophenyl-β-D-glucopyranoside,andforcellobiose7.
9mMand120U/mgunderoptimalconditions.
Theeffectsofthesubstrate,cel-lobiose(290mM),ontheenzymeactivitywerenotsignifi-cant.
TheKcat/Kmvalueforcellobiose13.
3mM-1s-1waslessthantheβ-glucosidasefromA.
oryzae,buttheactivityofβ-glucosidasefromA.
oryzaewasinhibitedbycellobiose,andrapidlydecreasedabove50°C(Table2).
Furthermore,theenzymeactivitywasenhancedbytheconcentrationsofglucosebelow200mM,andtheenzymeactivitywasFigure4TheeffectsofpHandtemperatureontheactivityandstabilityoftherecombinantBGL.
aEffectofpHonBGLactivity.
bEffectoftemperatureonBGLactivity.
cThepHstabilityoftheenzyme.
dThethermostabilityoftheBGL.
Theresidualactivitywasmonitored,whiletheenzymewasincubatedat50°C(filleddiamonds),65°C(filledsquares),68°C(filledtriangles),and70°C(letterx).
Theinitialactivitywasdefinedas100%.
Figure3SDS-PAGEanalysisofrecombinantBGLinE.
coliJM109(DE3).
LaneM:proteinmarker,lane1:cell-freeextractofJM109(DE3)harboringpET-20b,lane2:cell-freeextractofJM109(DE3)harboringpET-20b-BGL,lane3:cell-freeextractofJM109(DE3)harboringpET-20b-BGLII,lane4:purifiedBGL(4μg).
Table1EffectsofcationsandreagentsonpurifiedBGLactivityCationofreagentaResidualactivity(%)Control100Fe2+172Mg2+104Zn2+7Mn2+223Ca2+108K+101Al3+43Li+110Cu2+2Hg2+0Co2+37Ag2+19EDTA(10mM)102aFinalconcentration,1mMorasindicated.
Valuesshownarethemeanofduplicateexperiments,andthevariationaboutthemeanwasbelow5%.
Peietal.
BiotechnologyforBiofuels2012,5:31Page4of10http://www.
biotechnologyforbiofuels.
com/content/5/1/31increased110%whenadding100mMglucoseintoreactionmixtures(Figure5).
Whenglucosewasincreased,theen-zymeactivityofBGLwasgraduallyinhibited,withaKiof600mMglucose(Figure5).
Thepropertiesoftheglucose-tolerantβ-glucosidasefromothermicroorganismsaresum-marizedinTable2.
AsTable2shows,theseenzymeshavemanydistinctfeatures,especiallyintheircatalyticproper-ties[12,13,18-21].
AnalysisofcellobiosedegradationProductionofglucosefrom290mMcellobiose(10%)bythepurifiedBGLwasexamined.
Evenifthefinalconcen-trationofglucoseinreactionreachedabout580mM,cellobiose(290mM)wasfoundtobedegradedcom-pletely(Figure6a,b).
Atthebeginningofthereaction,theKcatwas67.
7s-1withinonehourat60°Cwhichwasidenticaltothetheoreticalvalue.
Duringthewholedeg-radationprocess,theKcatwas28.
2s-1.
PhylogeniesanalysisofBGLTogaininsightsintotheevolutionaryrelationshipamongβ-glucosidases,weconstructedthephylogenetictreesof40candidatesequencesusingheNJmethodandtheMPmethodrespectively,bothsupportingalmostthesametopology.
Theresultsrevealedthepresencesoffivewell-supportedclades:CladeIIwasGH1β-glucosidasesfromfungi,andCladeIIIwastheGH3β-glucosidasesfrombacteria,andCladeIVwastheGH3β-glucosidasesTable2Characteristicsofglucose-tolerantβ-glucosidasesfromT.
thermosaccharolyticumDSM571andothermicroorganismsStrainKm(mM)Vmax(U/mg)Kiforglucose(mM)Cellobioseinhibition(%)Kcat/Km(mM-1s-1)forcellobioseOptimalTemp(°C)pNPGaCellobiosepNPGCellobioseT.
thermosaccharolyticum0.
637.
964120600Noeffect13.
370Unculturedbacterium[13]0.
3920.
450.
715.
51000NDb0.
6540Debaryomycesvanrijiae[18]0.
7757.
966884.
3439ND2.
4340A.
oryzae[19]0.
5571,0663531,3905036.
150A.
niger[12]21.
7ND124.
4ND543NDND55A.
tubingensis[10]6.
2ND28.
40.
32600NDND60Candidapeltata[21]2.
3661088.
5c1400Noeffect0.
1c50Scytalidiumthermophilum[20]0.
291.
6113.
274.
12>200ND1.
760apNPG:p-nitrophenyl-β-D-glucopyranoside.
bND:notdetermined.
cItwascalculatedbythedatabasedonthereference.
Figure5TheeffectsofglucoseonBGLactivity.
Influenceofglucoseonenzymeactivitywithp-nitrophenyl-β-D-glucopyranosideasthesubstrate.
Figure6AnalysisofcellobiosehydrolysedbyBGL.
aThin-layerchromatographyoftheproductsfromthereaction.
M1:cellobiose,M2:glucose,lane0.
5,1,2,3,4,5,6:cellobiose(290mM)incubatedwithBGL(1μg)fordifferenttimes.
btheconcentrationofglucoseanalysisbyHPLC.
Peietal.
BiotechnologyforBiofuels2012,5:31Page5of10http://www.
biotechnologyforbiofuels.
com/content/5/1/31fromfungi.
TheGH1β-glucosidasesfrombacteriawasdividedintotwoclades:CladeImainlycontainedmeso-philicbacteria;CladeVmainlycontainedthermophile,whichisformedbyfurtherdividedintotwosubclades,ofwhichonecontainsallthermophile,andtheotherBacil-lusGH1β-glucosidases.
CladeIIandcladeIIIhadarela-tivelycloserelationship,andtheGH1β-glucosidasesfromthermophileweredistantfromtheotherclades(Figure7).
DiscussionsAclassificationofglycosidehydrolasesbasedonaminoacidsequencesimilaritieswasproposedafewyearsago,whereinβ-glucosidasesweremainlygroupedintotwosuperfamiliesofglycosidehydrolasesI(GH1),andGH3[22].
Although,theaminoacidsequenceanalysisindicatedthatBGLbelongstoGH1,itsharedthehighestsequencesimilarityof66%withtheβ-glucosidsesfromThermoanaerobactermathranii(YP_003676178.
1).
Moreover,itsharedonlythe63%withtheputativeβ-glucosidase(YP_004471891.
1)theThermoanaerobacteriumxylanolyticumLX-11,bothbelongingtothegenusThermoanaerobacterium.
ThePhylogeniesanalysisshowedthattheBGLwasdistantwiththeglucose-tolerantβ-GlucosidasesfromfungiandADD96762.
1(Figure7).
TheresultsindicatedthattheBGLcouldbeanovelβ-glucosidewithsomedifferentproper-ties.
Ontheotherhand,β-Glucosidasesmaybedividedintothreegroupsonthebasisoftheirsubstratespecificity.
Thefirstgroupisknownasaryl-β-glucosidasesowingtostrongaffinitytoaryl-β-glucose.
Thesecondgroupcon-sistsofcellobiasesthathydrolyzeoligosaccharidesonly.
Thethirdgroupisbroadspecificβ-glucosidasesthatexhibitactivityonawiderangeofsubstrates,andarethemostcommonlyobservedformofβ-glucosidases[23].
TheBGL,whichwashighaffinitytop-nitrophenyl-β-D-glucopyranoside,hydrolyzedcellobiose,p-nitrophenyl-β-D-glucopyranoside,andp-nitrophenyl-β-D-galactopyranoside,butnotp-nitrophenyl-α-L-arabinofuranoside,p-nitrophenyl-β-D-xylopyranoside,maltose,sucrose,andCMC.
TheseresultsindicatedthatBGLbelongedtothefirstgroup.
Enzymatichydrolysisofcelluloseisacomplexprocess,thelaststepbeingahomogenouscatalysisreactionFigure7TheNeighbor-Joining(NJ)andMaximum-Parsimony(MP)treesresultsfromanalysisofβ-glucosidasesof40aminoacidsequences.
Numbersonnodescorrespondtopercentagebootstrapvaluesfor1000replicates.
Peietal.
BiotechnologyforBiofuels2012,5:31Page6of10http://www.
biotechnologyforbiofuels.
com/content/5/1/31involvingtheactionofβ-glucosidaseoncellobiose.
Cellobioseisastronginhibitorofbothcellobiohydro-lasesandendocellulases.
Therefore,β-glucosidasewithhightoleranceforglucosehasbecomeheatedinthesefields.
Fungi,especiallyAspergillusspecies,aregenerallyconsideredtobeagoodproducerwithhighyieldofβ-glucosidases[24].
Butthemajorβ-glucosidasesbelong-ingtofamily3oftheglycosidehydrolases(GH3)fromAspergillusspeciesweresubjecttocompetitiveinhibitionofglucosetoproduceglucose,theKiisgenerally1–20mM[10,14].
Theminorβ-glucosidases,whichmolecu-larweightsare40–50kDa,exhibitedatolerancetoglucose(Table2).
TheeffectofglucoseontheBGLactivityrevealedthattheenzymeisnotonlyresistanttoend-productinhib-ition,butisactivatedbyglucoseatconcentrationsfrom0to0.
2M.
Onlytwoβ-glucosidases,activatedbyglucose,havebeenreportedfromScytalidiumthermophilumandmarinemicrobial(Table2)[13,20].
Moreover,highspecificactivityforcellobioseandtol-erancetosubstrateinhibitionareotheradvantagesforβ-glucosidaseinenzymatichydrolysisofcellulose.
Al-though,severalβ-glucosidasesfromafewfungiandbac-teriashowhighglucosetolerantwithKivaluesofmorethan200mM,theVmaxvaluesoftheseenzymesforcel-lobioseweremuchlowerthanforp-nitrophenyl-β-D-glucopyranoside.
TheVmaxvalueofBGLforcellobiosewas120U/mg,whichwasabout2timeshigherthantheVmaxvalueofBGLforp-nitrophenyl-β-D-glucopyranoside.
Toourknowledge,inonlyoneotherstudyhaveworkersdescribedthepurificationandcharacterization(fromA.
oryzae)ofaβ-glucosidasehavingsuchahightolerancetoglucoseandhighspecificactivityforcellobiose[19].
Butthespecificactivityofβ-glucosidasefromA.
oryzaeforcellobiosewasmuchlowerthanforp-nitrophenyl-β-D-glucopyranoside(Table2).
TheBGLwasonlytheβ-glucosidasebeenreportedthatitisnotonlyresistanttoglucose,buthadhigherspecificactivityforcello-biosethanforp-nitrophenyl-β-D-glucopyranoside.
Inaddition,theBGLhadhightolerancetosubstrateinhib-ition,cellobiose.
TheKcatofBGLwas67.
7s-1at60°CandpH6.
4,whentheconcentrationofcellobiosewas10%(Table2).
ThechemicalagentshadvariouseffectsontheactivityofBGL.
ThechelatingagentEDTAdisplayednoinflu-enceontheβ-glucosidaseactivity,indicatingthattheβ-glucosidaseisnotametalloprotein.
However,theβ-glucosidaseactivitywasgreatlystimulatedbyFe2+orMn2+,whichimpliedthatFe2+orMn2+isrequiredforthemaximalactivityofBGL.
TheseresultsdistinguishBGLfromtheotherbacteriaβ-glucosidases,onwhichCa2+showpositiveeffects[13].
Inpracticalapplications,thehighthermostabilityoftheenzymeisdesiredbecausethelongeractivelifemeansthelessconsumptionoftheenzyme.
TheBGLresidualactivitywasmorethan80%afterbeingincubatedat60°Cfor2h,anditinenzymatichydrolysisofcelluloseexhibitedhighactivityinbroadtemperature,whichcouldkeepathighlevelsattempera-turesfrom45to70°C.
ThepropertiesoftheBGLdemonstratedagreatpo-tentialofthegeneinthegeneticmodificationofstrainsforbiomassdegradation.
Differencesincodonusagepre-ferenceamongorganismsleadtoavarietyofproblemsconcerningheterologousgeneexpression,whichcanbeovercomebyrationalgenedesignandgenesynthesis.
Proteinwithmultiplerepetitiverarecodonsespeciallywithinthefirst20aminoacidsoftheaminoterminusoftheproteinmaysignificantlyreducetheproteinexpres-sion.
Sometimes,itshutsdowntheexpressioncom-pletely.
Sincetherarecodonsofbglfrom1–20aminoacidswereallchangedintooptimizedcodons,theactiv-ityofBGLwasincreasedbyabout70%(Figure3).
MoreoptimizationofcodonsfortheotheraminoacidresiduesintheORFofbglmaygivefurtherimprovementinthegeneexpressionlevels.
ConclusionWiththisstudy,wesuccessfullyover-expressedthenovelβ-glucosidase(BGL)genebglfromT.
thermosaccharoly-ticumDSM571byreplacingtherarecodonswiththeoptimalcodonsinE.
coli.
ThePhylogeniesanalysisshowedthattheBGLhadcloserelationshipwiththeβ-Glucosidasesfromthermophile,andwasdistantfromtheotherglucose-tolerantβ-Glucosidases.
Ascomparedontheenzymeproperties,theBGLwashighertoleranttoglucoseandcellobiose,moreefficientinhydrolysisofcellibiose,morethermalstabilitythanβ-glucosidasesfromothermicroorganisms.
Thus,thisstudyprovidesausefulnovelβ-glucosidase,whichmaybeusedtoim-provetheenzymaticconversionofcellulosictoglucosethroughsynergeticaction.
MaterialsandMethodsBacterialStrains,Plasmids,GrowthMediaThermoanaerobacteriumthermosaccharolyticumDSM571waspurchasedfromDSMZ(www.
dsmz.
de).
Itwasgrownanaerobicallyat60°Casdescribedpreviously[17].
EscherichiacoliJM109andJM109(DE3)wasgrownat37°CinLuria-Bertanimedium(LB)andsupplementedwithampicillinwhenrequired.
TheexpressionvectorspET-20b(Novagen)wereemployedascloningvectorandexpressionvector.
DNAmanipulationDNAwasmanipulatedbystandardprocedures[25].
QIA-GENPlasmidKitandQIAGENMinEluteGelExtractionKit(Qiagen,USA)wereemployedforthepurificationofplasmidsandPCRproducts.
DNArestrictionandmodifi-cationenzymeswerepurchasedformTaKaRa(Dalian,Peietal.
BiotechnologyforBiofuels2012,5:31Page7of10http://www.
biotechnologyforbiofuels.
com/content/5/1/31China).
DNAtransformationwasperformedbyelectro-porationusingGenePulser(Bio-Rad,USA).
Site-directedmutagenesisofgenesandthemodificationoftheplasmidswereperformedbyinverse-PCRfollowedbyphosporyla-tionandself-ligationusingT4polynucleotidekinaseandT4DNAligase.
PlamidconstructionsTheβ-glucosidasegenebglwasamplifiedfromT.
ther-mosaccharolyticumDSM571genomicDNAbyPCRusingprimersbgl-1andbgl-2(Table3),thePCRpro-ductsweredigestedwithNdeIandXhoIandinsertedintopET-20batNdeIandXhoIsites,yieldingtheplas-midpET-20-BGL.
Inordertoimprovetheexpressionlevelofrecombin-antBGL,theinternalregionfrom1stto19thaminoacidsinopenreadingframeofbglwasmutatedinsitubyin-verse-PCRtoreplacetherarecodonswiththeoptimalcodonsofE.
coli;theprimersfortheinverse-PCRweredesignatedasbgl-3andbgl-4(Table3).
Inverse-PCRwithprimerswascarriedoutusingPyrobestwithpET-20-BGLastemplate,generatingtheplasmidpET-20-BGLII.
ExpressionandpurificationofBGLPlasmidspET-20-BGandpET-20-BGLIIweretransformedintoE.
coliJM109(DE3),andinducedtoexpressedrecom-binantBGLbyaddingisopropyl-β-D-thiogalactopyranoside(IPTG)tofinalconcentrationof0.
8mMatOD600about0.
7,andincubatedfurtherat30°Cforabout6h.
OnelitersoftherecombinantcellscarryingpET-20-BGLIIwereharvestedbycentrifugationat5,000gfor10minat4°C,andwashedtwicewithdistilledwater,resuspendedin50mLof5mMimidazole,0.
5mMNaCl,and20mMTris–HClbuffer(pH7.
9),andFrench-pressuredforthreetimes.
Thecellextractswereheattreated(60°C,30min),andthencooledinanicebath,andcentrifuged(20,000g,4°C,30min).
Theresultingsupernatantswereloadedontoanimmobi-lizedmetalaffinitycolumn(Novagen,USA),andeludedwith1Mimidazole,0.
5MNaCl,and20mMTris–HClbuffer(pH7.
9).
ProteinwasexaminedbySDS-PAGE[26],andtheproteinbandswereanalyzedbydensityscanningwithanimageanalysissystem(Bio-Rad,USA).
ProteinconcentrationwasdeterminedbytheBradfordmethodusingBSAasastandard.
DeterminationofenzymeactivitiesandpropertiesThereactionmixture,containing50mMimidole-potassiumbuffer(pH6.
4),1mMp-nitrophenyl-β-D-glucopyranoside,andcertainamountofβ-glucosidasein0.
2mL,wasincubatedfor5minat70°C.
Thereac-tionwasstoppedbyadding1mLof1MNa2CO3.
Theabsorbanceofthemixturewasmeasuredat405nm.
Oneunitofenzymeactivitywasdefinedastheamountofenzymenecessarytoliberate1μmolofpNPperminundertheassayconditions.
TheoptimumpHforactivityβ-glucosidasewasdeter-minedbyincubationat70°Cfor5mininthe50mMimidole-potassiumbufferfrompH4.
8to8.
4.
Theoptimumtemperaturefortheenzymeactivitywasdeter-minedbystandardassayrangingfrom45to85°Cinthe50mMimidole-potassiumbuffer,pH6.
0.
TheresultswereexpressedaspercentagesoftheactivityobtainedateithertheoptimumpHortheoptimumtemperature.
ThepHstabilityoftheenzymewasdeterminedbymeasuringtheremainingactivityafterincubatingtheen-zyme(0.
1μg)at50°Cfor1hinthe50mMimidole-potassiumbufferfrompH5.
2to8.
0.
TodeterminetheeffectoftemperatureonthestabilityofBGL,theenzyme(0.
1μg)inthe50mMimidole-potassiumbuffer(pH6.
4)waspre-incubatedforvarioustimesat50°C,65°C,68°Cand70°Cintheabsenceofthesubstrate.
Theactivityoftheenzymewithoutpre-incubationwasdefinedas100%.
Theeffectsofmetalsandchemicalagentsonβ-glucosidaseactivityofpurifiedenzyme(0.
1μg)weredetermined.
Fe2+,Mg2+,Zn2+,Mn2+,Ca2+,K+,Al3+,Li2+,Cu2+,Co2+,andHg2+wereassayedatconcentra-tionsof1mMinthereactionmixture.
ThechemicalagentsEDTA(10mM)wereassayed.
Theenzymewasincubatedwitheachreagentfor10minat50°Cbeforeadditionofp-nitrophenyl-β-D-glucopyranosidetoiniti-atetheenzymereaction.
Activitywasdeterminedasdescribedaboveandwasexpressedasapercentageoftheactivityobtainedintheabsenceofthechemicalagentsandmetalcations.
Thesubstratespecificityoftheenzyme(0.
1μg)wastestedbyusingfollowingp-nitrophenyl-β-D-glucopyranoside,p-nitrophenyl-β-D-xylopyranoside,p-nitrophenyl-α-L-arabinofuranoside,maltose,sucrose,andcellobiose.
KineticconstantofBGLwasdeterminedbymeasuringtheinitialratesatvariousp-nitrophenyl-β-D-glucopyranosideconcentrations(0.
2,0.
4,0.
6,0.
8,1,2,and4.
0mM)orvariouscellobioseconcentration(2,4,6,8,10,12,14,and16mM)understandardreactioncondi-tions.
TheKivalueofglucosewasdefinedasamountofglucoserequiredforinhibiting50%oftheβ-glucosidaseTable3NucleotidesequencesofusedprimersPrimerNucleotidesequencebgl-1CCCCATATGTCGGACTTTAACAAGGACbgl-2CCCCTCGAGAATGGTCCTAGTGGAAATAAGbgl-3TTTGGCGTGGCGACCGCGAGCTATCAGGTGGAAGGTGCTTACAATGAGGAbgl-4CAGAAAATCTTTGTTAAAATCGCTCATATGTATATCTCCTTCTTAAAGTheboldfaceitalicnucleotidesrepresentedmutationsforoptimizingcodons.
Peietal.
BiotechnologyforBiofuels2012,5:31Page8of10http://www.
biotechnologyforbiofuels.
com/content/5/1/31activityandwasgivenastheaveragesofthreeseparateexperimentsperformedinduplicate.
PhylogeniesanalysisofBGLThecondonusagepreferenceofE.
coliintranslationiniti-ationregionofpET-20-BGLwasanalyzedbyusingcodonusagetool(http://gcua.
schoedl.
de/).
ThepotentialORFofbglwassearchedusingtheORFsearchtoolprovidedbytheNationalCenterforBiotechnologyInformation(www.
ncbi.
nlm.
nih.
gov).
DatabasesearchingwasperformedwithBlastatNCBIandagainstCAZy(www.
cazy.
org).
Theac-tivesiteoftheenzymewasanalyzedwiththeprositetool(http://prosite.
expasy.
org/scanprosite).
Themultiplese-quencealignmenttoolClustalX2.
0wasusedformultipleproteinsequencealignment[27].
Sequenceswerefurthereditedandalignedmanually,whennecessary,usingtheMega5forediting.
Forphylogeneticanalysesofconserveddomains,sequencesweretrimmedsothatonlytherele-vantproteindomainsremainedinthealignment[28].
PhylogeneticrelationshipswereinferredusingtheNeigh-bor-Joining(NJ)andMaximum-Parsimony(MP)methodasimplementedinPaup4.
0fortheNJandMPtrees,theresultswereevaluatedwith1000bootstrapreplicates[29].
ThegeneratedtreesweredisplayedusingTREEVIEW1.
6.
6(http://taxonomy.
zoology.
gla.
ac.
uk/rod/treeview.
html).
AnalysisofcellobiosedegradationThecellobiosewastreatedwithpurifiedBGL,andthedegradationwassubjectedtoanalysisofthin-layerchro-matography(TLC)andHPLC.
Thereactionmixture(20μL)contained290mMcellobiose,and1μgofBGLin50mMimidole-potassiumbuffer(pH6.
4).
Thereactionwasperformedforvarioustimesat60°C,andstoppedbyheatingfor5mininaboilingwaterbath.
Aftercentri-fugedfor10minat10,000g,supernatantsofthereac-tionmixtureswereappliedonsilicagelTLCplates(60F254,MerckCo.
).
Sugarsontheplateswereparti-tionedwithasolventsystemconsistingofn-butanol,aceticacid,andwater(2:1:1,byvol/vol),anddetectedusingtheorcinolreagent[30].
Theconcentrationofglu-cosewasexaminedbyHPLConacarbohydrateanalysiscolumn(WatersSugarpak1,USA)withwaterasamobilephase.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
AcknowledgementsThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(GrantNo.
31070515and30871990)andAProjectFundedbythePriorityAcademicProgramDevelopmentofJiangsuHigherEducationInstitutions(PAPD).
Authors'contributionsJPcarriedoutthecloningandover-expressionanddraftedthemanuscript.
QPandSFhelpedtopurifyandcharacterizetheBGL.
LZdirectedtheover-allstudyanddraftedthemanuscript.
HShelpedtoperformphylogeniesanalysisofβ-glucosidases.
Allauthorsreadandapprovedthefinalmanuscript.
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:Thermoanaerobacteriumthermosaccharolyticumβ-glucosidase:aglucose-tolerantenzymewithhighspecificactivityforcellobiose.
BiotechnologyforBiofuels20125:31.
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