A comparative study: Immobilization of yeast cells and invertase in poly (ethyleneoxide) electrodes

(1)

ContentslistsavailableatSciVerseScienceDirect

Journal

of

Molecular

Catalysis

B:

Enzymatic

j o ur na l h o me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / m o l c a t b

A

comparative

study:

Immobilization

of

yeast

cells

and

invertase

in

poly(ethyleneoxide)

electrodes

Huseyin

Bekir

Yildiz

∗

,

Musa

Kamaci,

Hacer

Azak,

Omer

Secgin,

Ozlem

Suer

DepartmentofChemistry,KaramanogluMehmetbeyUniversity,70100Karaman,Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14November2012

Receivedinrevisedform27January2013 Accepted26February2013

Available online 5 March 2013 Keywords: Yeastcells Invertase Immobilization Electropolymerization Conductingpolymers

a

b

s

t

r

a

c

t

Yeastcells(Saccharomycescerevisiae)andinvertaseenzymewereimmobilizedinthiophenecapped poly(ethyleneoxide)/polypyrrole(PEO-co-PPy)and3-methylthienylmethacrylate-co-p-vinylbenzyloxy poly(ethyleneoxide)/polypyrrole(CP-co-PPy)matrices.Immobilizationoftheenzymeandyeastcellswas performedviaentrapmentinconductingcopolymersduringelectrochemicalpolymerizationofpyrrole throughthethiophenemoietyofthepolymers.Maximumreactionrates,Michaelis–Mentenconstants, optimumtemperatureandpHvalues,operationalandstoragestabilitiesoftheenzymeandyeastcell electrodeswereinvestigated.

1. Introduction

Abiosensorisgenerallydeﬁnedasananalyticaltoolthatpairsa

biologicalcomponentwithadetectionsystemtoproducea

measur-ableresponse,whichcanbequantiﬁedintotheamountofanalyte

presentinasample[1].Theresponseisoftenmeasured

electro-chemicallyoroptically.Thetypesofbiologicalcomponentsthat

canbeusedareenzymes,antibodies,proteins, nucleicacids,or

wholecells.Wholecellsarelessexpensivetousebecausetheir

componentsrequirenoisolationandpuriﬁcation,asinthecell

com-ponentassaysystems.Also,wholecellsprovideinformationonthe

bioavailabilityofthetoxinswithinthesampleinsteadofsimply

reportingtheconcentrationpresent[2].

Immobilizationofsuchbiologicalagentsas,forinstance,

macro-molecules,cellsandmicroorganisms,iscommonlyproposedasa

successfultechnicalanswertotheincreasingdemandoflong-term

bio-efﬁciency.Itmayalsobeclaimedthatthisapproachprovides

controlled operationconditions withmaintenance of functions

andselectivity propertoa freebiological load[3].

Immobiliza-tionofcells containingspeciﬁcenzymeshasfurtheradvantages

suchaseliminationoflongandexpensiveproceduresforenzyme

separationandpuriﬁcation.Thewholecellenzyme

immobiliza-tionmethodgiveshighrecoveryofenzymeactivitycomparedto

theimmobilizationofpuriﬁedenzyme[4].Immobilizedmicrobial

∗ Correspondingauthor.Tel.:+903382262000;fax:+903382262116. E-mailaddresses:[email protected],[email protected](H.B.Yildiz).

cellderivatives are widelystudied inbiomedical,

pharmaceuti-cal and medical areas. Microbial cells have been immobilized

in several materials such as alginates [5–8], ceramics or glass

beads[9],cellulosicmaterials[10],␥-alumina[11],sol–gelsilica

ﬁlms[12],polyacrylamidegels[13]andconductingpolymers[14].

Theimmobilizationofyeastcellsexhibitinginvertaseactivityhas

beenperformedbyemployingCa-pectategels,ﬁlmsofpoly

(2-hydroxyethylmethacrylate),Ca-alginategels,liquid-corealginate

capsules,agaragarandwoolascarriermaterials[14].

Baker’syeast(Saccharomycescerevisiae)producesmany

impor-tantenzymes,whichareusednotonlyinthefoodindustry(mainly

infermentationprocesses)butalsoinchemicalsynthesis.Baker’s

yeastisaneconomicallyattractivebiocatalystduetoits

availabil-ityandlowcost,easeofhandlinganddisposal,safetyforfoodand

pharmaceuticalapplicationsaswellasitscapabilitytocatalyzea

widerangeofstereoselectivereactions.Itisnoteworthythat

reac-tionscarriedoutinthepresenceofbaker’syeastarepro-ecological

andmostofthemﬁtwithintheconceptofgreenchemistry[15].

Yeastcellsarewidelyusedinindustryduetoexhibitinggood

inver-tase activity.Invertase, ␤-fructofuranosidase(EC 3.2.1.26) is an

importantenzymeinthefoodindustryduetolowcost,good

sta-bilityandhighspeciﬁcactivity[16].Itcatalysesthehydrolysisof

sucroseintoanequimolarmixtureofglucoseandfructose,known

asinvertsugar.Invertsugarobtainedbythisenzymereactionand

itiscolourlessandhasahigheryieldofconversionthanthe

prod-uctthatisobtainedbyacidhydrolysis[17].Invertsugarhasalower

crystallinitythensucrose,whichisimportantinthefoodindustry

duetotheproductsremainfreshandsoftforalongtime.Invertase

isalsoimportantintheproductionofhighfructosesyrup[18].Also,

(2)

C

O

CH

2

S

m

Scheme1.StructureofCP-co-PPyconductingcopolymer[47,48].

invertasehasfoundmanyapplicationssuchasnon-crystallizable

sugar[19],biochemicalsensors[20],biopolymermembrane[21],

andcompositehydrogel[22].

Conductingpolymersserveasanappropriatematrix

supply-ingprevalentpropertiesforbiomoleculeimmobilization[23].Their

compatibility withbiological molecules, easy preparation, high

reproducibilityandelectrochemicalpropertiesmakethem

fasci-natinginbiosensordesign [24–26].Thesematerialscanalsobe

usedinmanyareas,suchaselectrochromicmaterials[27],

organic-based solarcells [28,29], organicﬁeld effect transistors [30,31]

andorganiclight-emitting[32,33].Conductingpolymersexhibit

awell-organizedmolecularstructuresothattheyhavethe

abil-itytofunctionasathreedimensionalmatrixforimmobilizationof

biomolecules[34–37].

In this study, yeast cells and puriﬁed invertase enzyme

were immobilized in 3-methylthienyl

methacrylate-co-p-vinylbenzyloxypoly(ethyleneoxide)/polypyrrole(CP-co-PPy)and

thiophenecappedpoly(ethyleneoxide)/polypyyrole(PEO-co-PPy)

matrices (Schemes 1–3). CP(3-methylthienyl

methacrylate-co-p-vinylbenzyloxy poly(ethyleneoxide), PEO (thiophene capped

poly(ethyleneoxide))andtheirconductingcopolymersofpyrrole

andthiopheneweresynthesizedandcharacterizedintheprevious

studies[38,39].Themaingoalofthestudyistoentrapthewhole

cellsinthepoly(ethyleneoxide)typeconductingpolymersinstead

of theinvertase enzyme toproduce invert sugar. The previous

studiesrelatedwithimmobilizationofwholecellswerefocusing

on the production properties and bioreactor parameters [14].

However, principal enzyme functions of immobilized whole

cells (kineticparameters, optimum reactiontemperatures, etc.)

werenotconsideredproperlyintheseimmobilizedsystems.This

work,therefore,aimstoclarifytheenzymaticparametersofthe

immobilizedyeastcellsintermsoftheenzymeinvertase.Thus,if

theimmobilizedwholecellscanshowacomparableactivitytothe

S CH2CH2 OH N H S _N N N N O H H H H H n CH2CH2 n H n

Scheme2.StructureofPEO-co-PPyconductingcopolymer[39,48].

pureinvertase,itwillbemorefeasibletoentrapthewholecells

andthenonedoesnothavetopurifythematerialtoobtainthe

pureinvertase.Byfollowingthesepurposes,optimumconditions

suchaspH,temperatureandkineticparameters(KmandVmax)

forimmobilizedyeastcellsandinvertaseinPEO-co-PPyand

CP-co-PPyconductingpolymers,wereinvestigated.Theoperationaland

storagestabilitystudiesoftheseenzymeandyeastcellelectrodes

werealsostudied.

2. Materialsandmethods

2.1. Materials

Baker’syeast (Saccharomycescerevisiae) waspurchased from

PakMaya(Turkey)andusedwithoutfurtherpuriﬁcation.Invertase

(EC3.2.1.26)TypeVandtannicacidwerepurchasedfromSigma

andusedasreceivedwithoutfurtherpuriﬁcation.Pyrrole(Merck)

wasdistilledbeforeuseandstoredat4◦C.Sodiumdodecylsulfate

(SDS)wassuppliedfromMerck.InordertoprepareNelsonreagent,

sodium carbonate, sodium potassium tartarate, sodium

bicar-bonate,sodiumsulfate,coppersulfatepentahydrate,ammonium

heptamolybdatetetrahydrate ((NH4)6Mo7O24·4H2O) and sodium

arsenate(Na2HAsO4·7H2O)wereprovidedfromAldrich.

2.2. Instrumentation

Potentioscan Wenking POS-73 and ST-88 potentiostats,

Shi-madzuUV-160-A modelspectrophotometer andJEOL JSM-6400

modelscanningelectronmicroscope(SEM)wereusedfor

charac-terization.

2.3. ImmobilizationofyeastcellsandinvertaseinPPyand

CP-co-PPymatrices

ImmobilizationofinvertaseinCP-co-PPymatrixwasachieved

byelectropolymerizationofpyrroleonapreviouslyCPcoated

plat-inumelectrode(1cm2_)._Enzyme_electrode_was_prepared_in₁₀_mL

acetatebuffer(pH5.0)containing1.2mg/mLSDSsupporting

elec-trolyte, 0.01Mpyrroleand 0.6mg/mL invertase.Immobilization

wascarriedoutataconstantpotentialof1.0Vfor20minatroom

temperature.Enzymeelectrodeswerekeptat4◦Cinacetatebuffer

solutionwhennotinuse.Fortheimmobilizationofyeastcellsin

CP-co-PPymatrix,theprocedureoftheimmobilizationoftheyeast

(3)

Scheme3.Schematicrepresentationofimmobilizationofyeastcellsinconductingcopolymermatrices.

1mg/mLofyeastcellsinsteadof0.6mg/mLinvertase.Enzyme

elec-trodeswerekeptat4◦Cin50mMacetatebuffersolution(pH5.0)

whennotinuse.

For theimmobilizationof invertasein PEO-co-PPy matrix, a

solutionof0.6mg/mLinvertase,2mg/mLPEO,1.2mg/mL

suppor-tingelectrolyte(SDS), 0.01Mpyrrole and10mL50mM acetate

buffer(pH5.0)wasputinatypicalthreeelectrodecell.Three

elec-trodecellhasthePtworkingandcounterelectrodesandaAg/Ag+

(0.01M)referenceelectrode.Immobilizationwascarriedoutata

constantpotentialof1.0Vfor20minatroomtemperature.Forthe

immobilizationofyeastcellsinPEO-co-PPymatrix,theprocedure

oftheimmobilizationoftheyeastcellswasthesameas

immobiliza-tionofinvertaseexceptforusing1mg/mLofyeastcellsinsteadof

0.6mg/mLinvertase.Enzymeelectrodeswerekeptat4◦Cin50mM

acetatebuffersolution(pH5.0)whennotinuse.

2.4. Invertaseactivitydetermination

Determinationofimmobilizedandsolubleinvertaseactivities

wasperformedusingNelson’smethod[40]forbothmatrices.

Dif-ferentconcentrationsofsucrosesolutionswerepreincubatedfor

10minat25◦C.Then,enzymeelectrodewasplacedinsucrose

solu-tionsforspeciﬁcreactiontimes(2,4and6min).Afterremovingthe

electrode,1mLaliquotsweredrawnandaddedto1mLNelson’s

reagenttoterminatethereaction.Thetubeswerethenplacedin

boilingwaterbathfor20min,thentheywerecooledtoroom

tem-peratureand1mLarsenomolybdatereagentwasadded.Finally,

7mLofdistilledwaterwasaddedtoeachtesttubeandmixedby

vortexing.Aftermixing,absorbancesfortheblankandthe

sub-stratesolutionsweredeterminedat540nmwithadoublebeam

spectrophotometer.Oneunitofinvertaseactivitywasdeﬁnedas

theamountofenzymerequiredtorelease1␮molglucosefrom

sucroseperminuteatpH5and25◦C.

The invertaseactivities of both suspended and immobilized

yeastcellsweredeterminedbymeasuringtheinitialreactionrates

ofsucrose hydrolysisat pH5.0,at 50◦C. Nelsonmethodwhich

wasexplainedabove,wasusedtodeterminereducingsugar

con-centration.Toconvertthespectrophotometerreadingstoenzyme

activities,astandardglucosecalibrationcurvewasprepared.One

unitofyeastinvertaseactivitywasdeﬁnedastheamountofyeast

cells required to produce 1mmol of glucose from sucrose per

minuteatpH5.0,at50◦C.

2.5. DeterminationofoptimumpHandoptimumtemperature

Thereactiontemperaturewaschangedbetween10◦Cand80◦C

whilesucroseconcentrationwaskeptconstantatabout10Kmfor

eachsystem.ForpHoptimizationat25◦Cfortheinvertaseenzyme

andat50◦Cfortheyeastcells,thepHofthereactionwaschanged

betweenpH2andpH11whilesucroseconcentrationwaskept

constantatabout10Kmforeachsystem.Theactivitieswere

deter-minedaspreviouslydescribed.

2.6. Operationalandstoragestabilityexperiments

Operational stabilities of immobilized invertase and yeast

cells in polymer ﬁlms were tested (at optimum activity assay

conditions) by performing 40 activity assays in one day while

sucroseconcentrationwaskeptconstantatabout10Kmforeach

(4)

Fortheshelflifedeterminationoftheenzymeandyeastcell

elec-trodes,theactivitieswerecheckedeverydayforaweekandthen

onceinﬁvedaysthroughout40dayswhilesucroseconcentration

waskeptconstantatabout10Kmforeachsystem.

2.7. Proteindetermination

Proteindeterminationmeasurementswereperformedby

Brad-ford’s Method. During measurements, a solution of Bradford

reagentwaspreparedbymixingonevolumestocksolutionwith

fourvolumesofdistilledwater.

Forthepreparationofproteincalibrationcurve,bovineserum

albumin(BSA) wasused. Different concentrations of BSA were

prepared with1mLand 2mL of diluted Bradford reagent. The

absorbanceofthesesolutionswasmeasuredat595nm.

Since the protein entrapped in enzyme electrode could not

bemeasured,wemeasuredtheproteinamountinthe

electroly-sissolutionbeforeandaftertheelectrolysis.Thedifferencegives

amountofproteinentrappedintheenzymeelectrodeduringthe

electrolysis[41].

3. Resultsanddiscussion

3.1. Proteindeterminationforenzymeandyeastcellelectrodes

Solubleinvertaseenzymewasfoundtohaveaspeciﬁc

activ-ityof81.7␮mol/minmgprotein.Resultsofproteindetermination

experiment for CP-co-PPy/invertase and PEO-co-PPy/invertase

electrodeswere4.68and3.39␮gproteinrespectively.

It was also proved that yeast cells were not damaged or

burstduringelectrolysisbycheckingproteinandDNA

concentra-tionswithinthesolutionsbothbeforeandafterblankelectrolysis

(without monomer) by means of absorbance measurements at

wavelengths280and260nm,respectively.Lackofsigniﬁcant

dif-ferencesintheproteinandDNAconcentrationsshowedthatyeast

cellswerenotburstandthecellmembraneswerenotbroken.The

spectrophotometerreadingsat600nmbeforeandafterelectrolysis

alsodidnotrevealanydifferenceinthecelldensity.

3.2. Kineticparametersofimmobilizedinvertaseandyeastcells

inCP-co-PPyandPEO-co-CPmatrices

Kinetic studiesof the immobilized invertase and yeast cells

were performed at constant temperature and pH while

vary-ingthesubstrateconcentration.Maximum velocity(Vmax) and

Michaelis–Menten constants (Km) for enzyme electrodes were

foundfromaLineweaver–Burkplot[42]whichisaplotof1/V0

against1/[S0]for systems obeyingtheMichaelis–Menten

equa-tion. The graph being linear can be extrapolated at anywhere

approximatingtoasaturatingsubstrateconcentration,evenifno

experimenthasbeenperformedandfromtheextrapolatedgraph,

thevaluesofKmandVmaxcanbedetermined.Michaelis–Menten

constant(Km)deﬁnestheafﬁnityofenzymetowardsitssubstrate.

LowertheKmvaluemeanshigheritsafﬁnityforthesubstrate.

Kineticconstantsforfreeandimmobilizedinvertasein

CP-co-PPyandPEO-co-PPymatricesaregivenin Table1.Thereaction

rates of the enzyme electrodes decreased becauseof the

difﬁ-cultyof formationof enzymesubstrate complex dueto porous

Table1

Kineticparametersforsolubleandimmobilizedinvertase.

Km Vmax

Freeinvertase 25.2mM 81.7␮mol/minmgprotein

PEO-co-PPy/invertase 16.3mM 4.71␮mol/mincm2

CP-co-PPy/invertase 19.4mM 3.28␮mol/mincm2

Table2

Kineticparametersforinvertaseactivityofsuspendedandimmobilizedyeastcells.

Km Vmax

Freeyeastcells 67.2mM 76.8␮mol/minmgprotein PEO-co-PPy/yeastcells 22.7mM 2.93␮mol/mincm2

CP-co-PPy/yeastcells 24.3mM 1.67␮mol/mincm2

structureofconductingpolymer.WhentheVmaxresultof

CP-co-PPyelectrodewascomparedwiththatofPEO-co-PPymatrices,it

wasseenthatVmaxvalueofCP-co-PPyelectrodewasthehigher

thanVmaxvalueofPEO-co-PPy.SinceVmaxvalueisdirectly

pro-portionaltotheamountofentrappedenzymeinthepolymer,these

resultsweresupportedbytheamountofproteinentrappedfor

invertaseenzymeintheelectrodesasdescribedabove.

Sucrase is another enzyme that catalyzes the hydrolysis of

sucrose to fructose and glucose and yeast cells, Saccharomyces

cerevisiae,mayhavesucraseandinvertasetogether.Tannicacid

andgallicacidareinhibitorsofthesucraseenzyme.Ifoneofthe

inhibitorsofsucraseenzymeisused,thecalculatedKmandVmax

willbelongtoonlyinvertaseactivityoftheyeastcells.Tobesure

thatwhethercalculatedVmaxandKmvaluesbelongtoinvertase

activityorsucraseactivityoftheyeastcellsforsucrosesubstrate,

enoughamountoftannicacidwhichistheinhibitorofthesucrase

enzyme wasused [43] andparallel resultswere obtainedwith

kineticconstantvaluesofpureinvertase.Table2showsthekinetic

constantsforinvertaseactivitybelongingtofreeandimmobilized

yeastcellsinCP-co-PPyandPEO-co-PPymatrices.TheKmvalue

forthesuspendedyeastcells wasobtainedas67.2mMsucrose.

0 2 4 6 8 10 12 0 20 40 60 80 100 120

R

el

ati

ve

A

cti

vi

ty

(%

)

pH

(a)

0 2 4 6 8 10 12 0 20 40 60 80 100 120

R

ela

tiv

e A

ctiv

ity

(%

)

pH

(b)

Fig.1. (a)EffectofpHonactivityofinvertaseimmobilizedinCP-co-PPy()and PEO-co-PPy( )matrices.(b)EffectofpHoninvertaseactivityofyeastcellsimmobilized inCP-co-PPy()andPEO-co-PPy( )matrices.

(5)

0 20 40 60 80 100 0 20 40 60 80 100 120

R

el

ati

ve

A

cti

vi

ty

(%

)

Temperature

(°C)

(a)

0 20 40 60 80 100 0 20 40 60 80 100 120

Temperature

(°C)

R

el

ati

ve

A

cti

vi

ty

(%

)

(b)

Fig.2.(a)Effectincubationtemperatureonactivityofinvertaseimmobilizedin CP-co-PPy()andPEO-co-PPy( )matrices.(b)Effectofincubationtemperatureon invertaseactivityofyeastcellsimmobilizedinCP-co-PPy()andPEO-co-PPy( ) matrices.

However,itwasobservedthattheKmvaluesofCP-co-PPy/yeast

cells and PEO-co-PPy/yeast cells electrodes are approximately

threetimeslowerthantheKmvalues ofsuspended yeastcells.

AlsoCP-co-PPy/yeastcellselectrodehashigherVmaxvaluethan

PEO-co-PPy/yeastcellselectrodedoes.ThelowKmindicateshigher

enzyme-substrate afﬁnity.The observed decreaseof Kmvalues

maybeconditionedbydiffusionallimitations.Besides,asmaller

KmvaluethansuspendedyeastcellsindicatesthattheCP-co-PPy

andPEO-co-PPymatrices providea microenvironmentwhich is

moresuitable thanthat inthesolution.Thesame behaviourof

thesematriceswasalsoseenfortheimmobilizedinvertase.

3.3. EffectofpHoninvertaseactivity

ChangesinpHcanaffecttheenzymestructureandalsocause

denaturation.Topreventthis, abuffersolutionshouldbeused.

In theseexperiments, thepH of thebufferwas 5.0. The

maxi-mumactivitywasobtainedatpH4.6[44]forthesolubleinvertase

enzyme.ThemaximumpHswerefoundtobe6.0forthe

CP-co-PPyand7.0forthePEO-co-PPymatrices.Theyareillustratedin

Fig.1a.Themaximuminvertaseactivityofsuspendedyeastcells

wasobtainedatpH5.0.Themaximuminvertaseactivityof

immo-bilizedyeastcells wereobservedatpH6.0 forthePPyandthe

CP-co-PPyand8.0forthePEO-co-PPymatrices.Theywereshown

inFig.1b.TheoptimumpHswereshiftedtowardsthealkalineside

whencomparedwiththesolubleenzyme.Thismightbeexplained

bypartitioningofprotons.Negativelychargedgroupsofthematrix

0 10 20 30 40 50 0 20 40 60 80 100 120

(a)

R

el

ati

ve

A

cti

vi

ty

(%

)

Assay Num

ber

0 10 20 30 40 50 0 20 40 60 80 100 120

(b)

R

el

ati

ve

A

cti

vi

ty

(%

)

Assay Num

ber

Fig. 3.(a) Operational stabilities of CP-co-PPy/invertase () and PEO-co-PPy/invertase( )electrodes.(b)OperationalstabilitiesofCP-co-PPy/yeastcells() andPEO-co-PPY/yeastcells( )electrodes.

willtendtoconcentrateprotons,andthiscausesloweringthepH

aroundtheenzyme. Therefore, thepHaround theenzymewill

belowerthanthat ofthebulkphase fromwhichthe

measure-mentofpHiscarriedout.WhilePEO-co-PPy/invertaseelectrode

lostonly 45% of maximuminvertase activity,PEO-co-PPy/yeast

cellselectrode lost35%ofinvertaseactivityofyeastcells upto

pH11(Fig.1b).Hence,this matrixcanbeusedreliablyat high

pHvaluesforenzymeandyeastcellreactions.Moreover,

CP-co-PPy/invertaseandCP-co-PPy/yeastcellselectrodesgavehighand

stableinvetaseactivitiesonthepHrangebetweenthepH3and

7andthismatrixcanbeusedinacidicmediasafely.Therelative

activitywascalculatedbyassigningthemaximumvalueofenzyme

activityas100%andthiswasdonealsofortemperature

optimiza-tion,operationalstabilityandstoragestabilityexperimentsofthe

biosensor.

3.4. Effectoftemperatureoninvertaseactivity

Themaximumtemperatureforthesolubleinvertaseenzyme

wasfoundto be50◦C [45] Both enzymeelectrodes revealed a

temperatureof 50◦C where invertaseshowed maximum

activ-ity.AlthoughtheCP-co-PPymatrixatlowtemperaturesshowed

higher stability, at high temperatures the enzyme activity for

both matrices reduces rapidly. The reason of rapid reduction

canbe explained asthat while thetemperature increases

con-tinuously after the optimum temperature, the structure of the

enzymebecomesalteredanditscatalyticpropertiesarereduced

(6)

0 10 20 30 40 50 0 20 40 60 80 100 120

(a)

R

el

ati

ve

A

cti

vi

ty

(%

)

Days

0 10 20 30 40 50 0 20 40 60 80 100 120

(b)

R

el

ati

ve

A

cti

vi

ty

(%

)

Days

Fig.4. (a)StoragestabilityofCP-co-PPy/invertase()andPEO-co-PPy/invertase( ) electrodes.(b)StoragestabilityofCP-co-PPy/yeastcells()andPEO-co-PPY/yeast cells( )electrodes.

the invertaseenzyme very wellat high temperatures.

PEO-co-PPy/invertaseelectrodelostonly25%ofinvertaseactivityupto

80◦C(Fig.2a).

Inapreviousstudyoptimumtemperatureforinvertaseactivity

ofsuspendedyeastwasfoundas55◦C[14].Fig.2bshowsrelative

percent invertase activitiesversus temperature in three

differ-entimmobilizationsystems,respectively.Themaximuminvertase

activityofyeastcells wasobservedat50◦CforCP-co-PPy/yeast

cellselectrode.Maximuminvertaseactivityofyeastcellsfor

PEO-co-PPyandmatrixwasseenat60◦C.Afterthemaximumpoint,

temperaturesupto80◦C,85%and55%ofmaximumactivitieswere

retainedinPEO-co-PPyandCP-co-PPyrespectively.

3.5. Operationalandstoragestabilityofenzymeandyeastcell

electrodes

Enzymescaneasilylosetheircatalyticactivityanddenatured,so

carefulstorageandhandlingofenzymesareimportant.The

CP-co-PPy/invertaseelectrodehadhighoperationalstabilitywhichwas

around85–90%ofmaximumenzymeactivityduringthe40

exper-imentsperformedat25◦C,onthedayofimmobilization.Inspiteof

havinggoodenzymeprotectionagainsthightemperatureandpH,

PEO-co-PPymatrixdidnotprotectenzymeverywelland

PEO-co-PPy/invertaseelectrodelost75%ofitsactivityuntil30thuseand

stayedconstantupto40thuse.Thestabilityofyeastcellelectrodes

intermsofrepetitiveuseswasstudiedbyperforming40successive

measurementsat50◦Cinoneday.AlthoughCP-co-PPy/yeastcells

electroderevealedanactivitythatgraduallydecreaseduntil35th

Fig.5.Scanningelectronmicrographsof(a)CP-co-PPymatrixwithyeastcellsand (b)PEO-co-PPymatrixwithyeastcells.

useandstayedconstantat80%ofitsactivity,PEO-co-PPy/yeastcells

electrodedidnotexhibitedhighoperationalstabilityandretained

45%ofitsactivityafter40thuse(Fig.3).Theslightincreaseinthe

responsesofCP-co-PPy/invertaseandCP-co-PPy/yeastcells

elec-trodesarerelatedtotheswellingofthepolymerstructureandit

wasspeculatedthatswellingofthepolymermaycausechanging

positionsoftheenzymemoleculesinthepolymertoincreasethe

enzymeactivityslightly[46].Asbeingknowthatduetophysical

entrapment,thereisnobondbetweenenzymesandthepolymer

inthepolymerandwhenthepolymerswells,positionchangingfor

enzymemoleculescanhappen.

Forthestoragestabilitiesoftheinvertaseenzymeandyeastcell

electrodes,theactivitieswerecharacterizedeverydayforaweek

and thenoncein 5daysthroughout 40daysat 25◦C and50◦C

respectively. Theinvertaseenzyme in CP-co-PPymatrice

main-tained75%ofitsactivityafter15daysandstayedconstantupto

40thday.PEO-co-PPy/invertaseelectrode maintained50%ofits

invertaseactivityafterabout20thdayandstayedconstantuntil

40thday.TheyeastcellsinCP-co-PPymatrixmaintained80%of

(7)

day.PEO-co-PPymatrixlost65%ofinvertaseactivityoftheyeast

cellsandstayedconstantuntil40thday(Fig.4).

3.6. Morphologiesofﬁlms

In order to determinethe surface morphologies of polymer

ﬁlms,scanning electronmicroscopy (SEM)technique wasused.

ScanningelectronmicrographsofCP-co-PPy/yeastcellsand

PEO-co-PPy/yeast cells electrodes are given in Fig. 5. The surface

propertiesofCP-co-PPyand PEO-co-PPymatriceswithoutyeast

cellsweregiveninpreviousstudies[38,39].Thesurface

morpholo-giesoftheseﬁlmswerecompletelydifferentcomparedtotheﬁlms

preparedintheabsenceofyeastcells.Cauliﬂower-likestructure

wasnoticeablydamagedwhenyeastcellswereentrappedinthese

matrices.

4. Conclusion

Inthisstudy,yeastcellswereusedasamodelforwholecell

immobilizationin poly(ethyleneoxide)type conductingpolymer

matricesandtheinvertaseactivityoftheimmobilizedcellswere

investigated. The immobilized cell system showed comparable

kineticdatawiththatoftheimmobilizedenzyme.Measurements

performedat25◦Candcomparisonwithpureinvertaserevealed

thatthesubstrateafﬁnityandmaximumreactionrateof

inver-taseimmobilizedinCP-co-PPyandPEO-co-PPYmatriceswasnot

changedwhetherpureinvertase oryeastcells wereused.

PEO-co-PPy/yeast cells and PEO-co-PPy/invertase electrodeshad the

smallestKmwhencomparedwithKmvaluesofsolubleenzyme,

suspended yeastcells, CP-co-PPy/invertaseand CP-co-PPy/yeast

cells electrodes.The smallestKmvalueshows that PEO-co-PPy

matrixprovidesamicroenvironmentwhichismoresuitablethan

thatinthesolutionandCP-co-PPymatrix.Itcanbeunderstoodthat

invertaseandyeastcellsimmobilizedinCP-co-PPymatrixbinded

theirsubstratemorestrictlythanthefreeorsolubleenzymeinthe

solutionandenzymeandyeastcellsimmobilizedinPEO-co-PPy

matrixdid.InspiteofhavinghightemperatureandpHstabilities,

PEO-co-PPy/yeastcellsandPEO-co-PPy/invertaseelectrodescould

nothavegoodoperationalandstoragestabilities.Thereasonofthe

badstabilitiescanbecommentedasthatthismatrixcouldnot

pro-tectyeastcellsandtheenzymewellandsomeofthemdroppedto

solutionfromthematrix.Asaresult,itcanbesaidthatthewhole

cellscanbeentrappedinsteadoftheinvertaseenzymeandthenone

doesnotneedtopurifythematerialtoobtainthepureinvertase.

Sinceinvertaseissuchacheapenzyme,itisusedratherinnative

formintheindustry.However,thisstudycanserveasamodelfor

theimmobilizationofmoreexpensiveenzymes,inwhichcasethe

usageofunpuriﬁedformwouldbeeconomical.

Acknowledgements

This study was supported ﬁnancially by the Scientiﬁc and

TechnologicalResearchCouncilofTurkey(TUBITAKGrant

Num-ber109T439)andtheScientiﬁcResearchProjectsFoundationof

KaramanogluMehmetbeyUniversity(KMU-BAPGrantNumber

09-M-11).Ms.SuerwouldliketothankScientiﬁcandTechnological

ResearchCouncilofTurkeyforitsSupportingResearchProjects

Pro-gramfortheUniversityStudentsatundergraduatelevel(Program

NumberTUBITAK-2209).

References

[1]S.Belkin,Curr.Opin.Microbiol.6(2003)206–212.

[2] K.A.Linares,Evaluatingstrategiesforintegratingbacterialcellsintoa biosen-sordesignedtodetectelectrophilictoxins.M.Sc.Thesis,VirginiaPolytechnic InstituteandStateUniversity,Blacksburg,Virginia,USA,2009,pp.12–13,Chap. 1.

[3] E.Callone,R.Campostrini,G.Carturan,A.Cavazzab,R.Guzzon,J.Mater.Chem. 18(2008)4839–4848.

[4] P.Brodelius,E.J.Vandamme,in:J.F.Kennedy(Ed.),Immobilizedcellsystemsin Biotechnology,Vol.7a,VCSVerlagsgeselleschaft,Germany,1987,p.40. [5] M.Fumi,G.Trioli,O.Colagrande,Biotechnol.Lett.9(1987)339–342. [6] I.Yoo,G.H.Seong,J.K.Park,EnzymeMicrob.Technol.19(1996)428–433. [7] E.Corton,M.Piuri,F.Battaglini,S.M.Ruzal,Biotechnol.Prog.16(2000)59–63. [8] A.Idris,W.Suzana,ProcessBiochem.41(2006)1117–1123.

[9] L.M.D.Goncalves,M.T.O.Barreto,A.Xavier,M.J.T.Carrondo,J.Klein,Appl. Microbiol.Biotechnol.38(1992)305–311.

[10]E.Bardi,A.A.Kountinas,J.Agric.FoodChem.42(2004)221–226.

[11]P.Loukatos,M.Kiaris,I.Ligas,G.Burgos,M.Kannelaki,M.Komaitis,A.A. Kouti-nas,Appl.Biochem.Biotechnol.89(2000)1–13.

[12]L.Inama,S.Dire,G.Carturan,A.Cavazza,J.Biotechnol.30(1993)197–210. [13] J.Rossi,F.Clementi,Am.J.Enol.Viticult.63(1984)100–102.

[14]Z.Balci,U.Akbulut,L.Toppare,S.Alkan,U.Bakir,Y.Yagci,J.Macromol.Sci.Pure 39(2002)183–197.

[15]I.Stolarzewicz,E.Bialecka-Florjanczyk,E.Majewska,J.Krzyczkowska,Chem. Biochem.Eng.Q.25(2011)135–144.

[16]H.Mohammadi,A.Amine,M.ElRhazi,C.M.A.Brett,Talanta62(2004)951–958. [17]T.Danisman,S.Tan,Y.Kacar,A.Ergene,FoodChem.85(2004)461–466. [18]E.J.Tomotani,M.Vitolo,J.FoodEng.80(2007)662–667.

[19] S.M.Kotwal,V.Shankar,Biotechnol.Adv.27(2009)311–322.

[20]D.Bagal-Kestwal,R.M.Kestwal,B.H.Chiang,M.S.Karve,Sens.ActuatorsB: Chem.160(2011)1026–1033.

[21]D.Bagal,M.S.Karve,Anal.Chim.Acta555(2006)316–321. [22] H.N.Oztop,C.Hepokur,D.Saraydin,J.FoodSci.74(2009)N45–N49. [23]M.Gerard,A.Chaubey,B.D.Malhotra,Biosens.Bioelectron.17(2002)345–359. [24]O.Turkarslan,S.Kayahan,L.Toppare,Sens.ActuatorsB:Chem.136(2009)

484–488.

[25] P.Camurlu,A.Cirpan,L.Toppare,Synth.Met.146(2004)91–97.

[26]S.Varis,M.Ak,C.Tanyeli,I.M.Akhmedov,L.Toppare,Eur.Polym.J.42(2006) 2352–2360.

[27]J.H.Tsai,C.C.Chueh,W.C.Chen,C.Y.Yu,G.W.Hwang,C.Ting,E.C.Chen,H.F. Meng,J.Polym.Sci.PartA:Polym.Chem.48(2010)2351–2360.

[28] G.Y.Chen,C.M.Chiang,D.Kekuda,S.C.Lan,C.W.Chu,K.H.Wei,J.Polym.Sci. PartA:Polym.Chem.48(2010)1669–1675.

[29]J.Song,C.Zhang,C.Li,W.Li,R.Qin,B.Li,Z.Liu,Z.Bo,J.Polym.Sci.PartA:Polym. Chem.48(2010)2571–2578.

[30] C.C.Chueh,M.H.Lai,J.H.Tsai,C.F.Wang,W.C.Chen,J.Polym.Sci.PartA:Polym. Chem.48(2010)74–81.

[31]M.A.M.Leenen,T.Meyer,F.Cucinotta,H.Thiem,R.Anselmann,L.DeCola,J. Polym.Sci.PartA:Polym.Chem.48(2010)1973–1978.

[32] H.Su,F.Wu,C.Shu,Y.Tung,C.Yun,G.Lee,J.Polym.Sci.PartA:Polym.Chem. 43(2005)859–869.

[33]M.Sendur,A.Balan,D.Baran,L.Toppare,J.Polym.Sci.Polym.Chem.49(2011) 4065–4070.

[34]F.R.R.Teles,L.P.Fonseca,Mater.Sci.Eng.C28(2008)1530–1543.

[35]Y.Xian,Y.Hu,F.Liu,Y.Xian,H.Wang,L.Jin,Biosens.Bioelectron.21(2006) 1996–2000.

[36]A.Ramanaviciene,W.Schuhmann,A.Ramanavicius,ColloidsSurf.B48(2006) 159–166.

[37]E.Baskurt,F.Ekiz,D.OdacıDemirkol,S.Timura,L.Toppare,ColloidsSurf.B97 (2012)13–18.

[38] H.B.Yildiz,S.Kiralp,L.Toppare,F.Yilmaz,Y.Yagci,K.Ito,T.Senyo,Polym.Bull. 53(2005)193–201.

[39]H.B.Yildiz,S.Kiralp,L.Toppare,Y.Yagci,K.Ito,Mater.Chem.Phys.100(2006) 124–127.

[40]N.Nelson,J.Biol.Chem.153(1944)375–380.

[41]S.Kiralp,L.Toppare,Y.Yagci,Int.J.Biol.Macromol.33(2003)37–41. [42]H.Lineweawer,D.Burk,J.Am.Chem.Soc.56(1934)658–666.

[43]S.Gupta,S.Mahmood,R.H.Khan,A.Mahmood,Biosci.Rep.30(2009)111–117. [44]N.Kizilyar,U.Akbulut,L.Toppare,M.Y.Ozden,Y.Yagci,Synth.Met.104(1999)

45–50.

[45]R.Erginer,L.Toppare,S.Alkan,U.Bakir,React.Funct.Polym.45(2000)227–233. [46]A.Cirpan,S.Alkan,L.Toppare,I.Cianga,Y.Yagci,Des.MonomersPolym.6

(2003)237–243.

[47] H.B.Yildiz,L.Toppare,Biosens.Bioelectron.21(2006)2306–2310.

[48]H.B. Yildiz,S.Kiralp,L. Toppare,Y.Yagci,React.Funct.Polym.63(2005) 155–161.