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Surfactant-coated

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Surfactant-coatedSurfac

    Surfactant-coated

ChineseJ.CheH].Eng.,11(5)601603(2003)

    ;RESEARCHNoTES

    ;SurfactantcoatedandidarugosaLipaseasCatalystfor

    ;HydrolysisofOliveOilinSolvent.Free

    ;TwoPhaseSystem

    ;SONGBaodong(宋宝东),,DINGHui(T),wuJinchuan(吴金川)a,

    ;HayashiY.,TalukderMMRand,vANGShichang(~世昌)a

    ;ChemicalEngineeringResearchCenter,SchoolofChemicalEngineeringandTechnology ;,TianjinUniversity,Tian

    ;jin300072,China

    ;DepartmentofChemistry&ChemicalEngineering,FacultyofEngineering,KanazawaUniversity2-4020Kodat

    ;suno,Kanazawa9208667,Japan

    ;AbstractThesurfactantcoatedCandldarugosalipasewasusedascatalystforhydrolysisofoliveoilintHphase

    ;systemconsistingofoliveoilandphosphatebufferwithoutorganicsolvent.Forboththecoatedandnativelipases.

    ;theoptimalbuffer/oilvolumeratioof1.0,aqueouspH6.8andreactiontemperature30~(2weredetermined.The

    ;maximumactivityotthecoatedlipasewasca1.3timesthanthatofthenativelipase.Thehalf-lifeofthecoated

    ;lipaseinoliveoilandthenativelipaseinphosphatebufferwasca9hand12handthefinalresidualactivitvwas

    ;27%and20%oftheirinitialvalues,respectively.Thefinalsubstrateconversionbythecoatedlipasewasca20%

    ;higherthanthatofthenativelipase.

    ;KeywordsCandidarugosa,hydrolysis,lipase,oliveoil,solventfreesystem,surfactant

    ;1INTRoDUCTIoN

    ;Enzymatichydrolysishasbeenregardedasa

    ;promisingwayforsplittingfatsandoils[Jowingtoits

    ;inherentadvantagessuchasthemildconditionsand

    ;eliminationofcolorimpurityovertheconventional

    ;processthatproceedsathightemperatureunderhigh

    ;pressure.However.theeconomiccompetenceofthe

    ;enzymaticprocessisdependent,toalargeextent,on

    ;theimprovementinactivityandthestabilityofbio

    ;catalystsaswellasthecostindownstreamseparation.

    ;Surfactantcoatedenzymeshavebeenextensively

    ;studiedaspromisingbiocatalystsforsynthesisre

;actionsinanhydrousorganicsolvents[241.

    ;Moriet

    ;a1.L5Jreportedforthefirsttimetheapplicationof ;surfactant.coatedenzymesashydrolyticcatalystfor ;lipophilicestersintwo--phasesystemsconsistingofor.. ;ganicsolventandaqueousbuffer.Asthesurfactant

    ;coatedenzymesissolubleinawiderangeoforganic ;solventsincludingsomepolaronesbutinsolublein ;aqueoussolutiont6J.itispossibletousethemasbio

    ;catalystsinasolvent.freesystemprovidedthatthe ;coatedenzymessolubilizeorat1eastwelldisperse ;inthereactionmediumformedbyorganicreactants ;alonesoastosimplifythedownstreamseparationin ;comparisonwiththatinthesolventcontainingsys.

    ;tems.Theobjectiveofthisworkistoinvestigatethe ;possibilityofusingthesurfactantcoatedCandaru

    ;gosalipaseasbiocatalystforhydrolysisofoliveonin ;asolventfreetwophasesystem.

    ;2MATERIALSANDMETHoDS

    ;2.1Materials

    ;Lipase(EC3.1.1.3)fromCandidarugosa(Type ;VII)waspurchasedfromSigma.Thenonionicsurfac

    ;tant,glutamicaciddidodecylesterribitolamide.used ;forcoatingthelipasewassynthesizedfollowingthe ;methodofGotoeta1.tr[.Oliveoilwithasaponifica

    ;tionvalueof195.0wasfromBeijingChemicalReagent ;Co..China.Aotherchemicalsusedwereofanalyti

    ;calgradeandobtainedcommercially.

    ;2.2Preparationofthesurfactantcoatedlipase

    ;Thesurfactantcoatedlipasewaspreparedaccord

    ;ingtothemethodofGotoeta1.6Jwithaslightmod

    ;ificationasdescribedinapublicationIs]. ;2.3Proteinassay

    ;Theproteincontentinthesurfactant.coatedlipase ;wasdeterminedbasedontheUVabsorptionofaro

    ;maticaminoacidresiduesinproteinsat280nmfol

    ;lowingtheproceduresofOkahataeta1._9J. ;2.4Determinationoflipaseactivityandsta

    ;bility

    ;Atypicalexperimentalprocedurewasasfollows. ;1.5mloliveoil(1.0mo1.L)containingsurfactant

    ;coatedlipase(O.55mgprotein)wasmixedwith3.0ml ;O.1mo1.LphosphatebuerofPH6.8.Themixture

    ;wasincubatedat3O.CfOr3Ominwithcontinuously

;stirringat250r.min1.Then10mlethanol/acetone

    ;(1:1yvolume)wasaddedtostopthereaction.The ;fattyacidsproducedweredeterminedbytitration ;Received2003O112.accepted20030507.

    ;SupportedbytheNationalNaturalScienceFoundationofChina(No.29876031)

    ;Towhomcorrespondenceshouldbeaddressed.Email:bdsong~tju.edu.cn ;

    ;602ChineseJ.Ch.E.(Vo1.11,No.5)

    ;with0.01mo1.LNa0H.Specificactivityoftheen. ;zymewasexpressedasmmolfattyacidsperminper ;mglipase.Forcomparison,thehydrolyticactivityof ;thenativeCandidarugosalipasewasalsoinvestigated ;underthesameconditions.

    ;Forinvestigationofenzymestability,thecoated ;lipaseinoliveoilorthenativelipaseinphosphate ;bufferwasincubatedat30.Cforpredeterminedtime ;intervalsandtheresidualactivitywasmeasuredas ;describedabove.

    ;3RESULTSANDDISCUSSION

    ;3.1Eff-ectofvolumeratioofphosphatebuffer ;tooliveoilonenzymeactivity

    ;Figure1showsthatboththesurfactantcoated

    ;andnativeCandidarugosalipasesreachedthemaxi

    ;mumatsomeintermediatevolumeratioofphosphate ;buffertooliveoil.Itisnoticedthattheactivityof ;thecoatedlipasewashigherthanthatofthen&- ;tivelipaseespeciallyatthemediumvolumeratios. ;Themaximumactivityofthecoatedlipasereached ;35.9mmo1.min_..g_..3O%higherthanthatoftheha- ;tivelipase.Thehigheractivityofthecoatedlipase ;mightbeascribedtothegoodsolubilizationofthe ;coatedlipaseinoliveoilfavoringthebiocatalysis. ;30

    ;20

    ;O0l2345

    ;buffer/oliveoil(byvolume)

    ;Figure1Effectofvolumeratioofoliveoilto ;phosphatebuffer(O.1mol?L_.,pH6.8)onspecific ;activityofthesurfactantcoated(?)andnative(O)

    ;Candidarugosalipases

    ;(oliveoil:1,5ml,lipase:O.55mg,reactiontemperature:30~C)

    ;3.2EfiectofbufferPH

    ;Figure2indicatesthattheactivityofboththe ;surfactant.coatedandnativelipaseswasstronglyde..

    ;pendentonthebufferpH,andtheoptimumpHwas ;around7.Asexpected,theactivityofthecoatedli

    ;pasewashigherthanthatofthenativelipaseatall ;pHlevels.

    ;3.3Efiectofreactiontemperature

    ;Figure3demonstratesthatboththecoatedand ;nativelipasesshowedmaximumactivityataround ;30oC.Abovethistemperaturetheactivityofthe ;coatedenzymedecreasedsmoothlyduringthewhole ;temperaturerangewhereasthenativelipasedeacti

    ;October,2003

    ;vatedrapidlyafter50~C.Asthecoatingofenzymes ;withsurfactantsisthroughhydrogenbondlinkages ;betweenthemanddoesnotaffecttheenzymeactive ;site[,theintrinsicpropertiesofthelipasesuchasthe ;optinmmreactiontemperaturewashardlyaffected. ;Figure2EffectofbufferpHonspecificactivityof ;thesurfactantcoated(?)andnative(O)Candida

    ;rugosalipases

    ;[oliveoil:1.5ml,phosphatebuffer(o.1tool?L):3.0ml,

    ;lipase:o.55mg,reactiontemperature:30~C] ;.0

    ;

    ;E

    ;?

    ;E

    ;E

    ;-

    ;;

    ;;

    ;temparature.?

    ;Figure3Effectofreactiontemperatureonspecific ;activityofthesurfactant-coated(?)andnative(0)

    ;Candidarugosalipases

    ;[oliveoil:1.5ml,phosphatebuffer(o.1mol?L_.,pH6.8) ;3.0ml,lipase:o.55mg]

    ;3.4Lipasestability

    ;Thestabilityofanenzymeisofgreatimportance ;foritscommercialapplications.Figure4depictsthat ;theactivitiesofthecoatedandnativelipasesrespec

    ;tivelydecreasedtoca27%and2O%oftheirinitial ;valuesat28h.Thehalf-lifeofthecoatedlipasein ;oliveoilwasca9h,whilethatofthenativelipasein ;phosphatebufferwasca12h,slightlyhigherthanthe

;former.However,consideringthehigherinitialactiv

    ;itvofthecoatedlipase.theabsoluteactivityofthe ;coatedlipasewashigherthanthatofthenativelipase ;duringthewholetimeperiod.

    ;3.5Timecourseofhydrolyticreaction

    ;Figure5displaysthatthesubstrateconversionin

    ;creasedrapidlywithtimewithinthefirst10handthe ;reactionbegantoslowdownafterwards,whichmight ;beascribedtoenzymedeactivation,productinhibi

    ;tionand/orlimitationbychemicalequilibrium.As ;

    ;_.一三E0EElll.

    ;.一三E0EEl1三芑

    ;

    ;Surfactant-coatedCandidar,ugosaLipaseasCatalystforHydrolysisofOliveOil603

    ;expected.thesubstrateconversionofthecoatedli. ;pasewashigherthanthatofthenativelipase.Forin. ;stance,thesubstrateconversionsrespectivelyreached ;41%and33%at32h,theformerwas1.2timeshigher ;thanthelatter.Therefore.thesurfactant.coatedCan. ;didarugosalipaseismoresuitableforcatalyzingthe ;hydrolysisofoliveoilinasolvent.freetwo.phasesys. ;ternthanthenativelipase.

    ;time.hREFERENCES

    ;Figure4Stabilityofthesurfactant-coated(?)and

    ;native(0)Candidarugosalipasesat30~Casa ;functionoftime

    ;fThecoatedlipasewasincubatedinoliveoilandthenative ;lipasewasincubatedinphosphatebufferfO.1mol?L,pH

    ;6.8).Assayconditions:oliveoil:1.5ml,lipase:O.55mg, ;phosphatebuffer(O.1mol?L,pH6.8):1.5mlforcoated

    ;lipaseand3.0nllfornativelipase.reactiontemperature:30.C ;Thei00%relativeactivitiesofthecoatedandnativelipases ;representtheactivityvaluesof35,9and27.1mmol?min?g

    ;respectively]

    ;time.h

    ;Figure5Timecourseofoliveoilhydrolysis

    ;catalyzedbythesurfactant-coated(?)andnative(O)

    ;Candidarugosalipases

    ;foliveoil:1.5ml,phosphatebuffer(O.1mol?L,pH6.8):1.5

    ;and3.0mlforthecoatedandnativelipases,respectively, ;lipase:O.55mg,reactiontemperature:30.C1 ;lWang,Y.J.,Shou,J.Y.,Wang,F.F.,Shaw,J.F.,”Lipase

    ;.

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    ;3Noda,S,,Kamiya,N.,Goto,M.,Nakashio,F.,”Enzymatic

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    ;5Mori,T.,Kishimoto,S.,Ijiro,K.,Kobayashi,A.,Okahata, ;Y.”Alipid—coatedlipaseasanefficienthydrolyticcata. ;1ystinthetwo-phaseaqueous—organicsystem”,Biotechno1.

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    ;maticesterificationbysurfactant-coatedlipaseinorganic ;media”,Biotechno1.Prog.,10(2),263268(1994).

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    ;9Okahata,Y.,Ijiro,K.,[标签:快照]

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