L-Dopa Synthesis Catalyzed by Tyrosinase Immobilized In Poly(ethyleneoxide) Conducting Polymers

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InternationalJournalofBiologicalMacromolecules56 (2013) 34–40

ContentslistsavailableatSciVerseScienceDirect

International

Journal

of

Biological

Macromolecules

j our 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 / i j b i o m a c

l-Dopa

synthesis

catalyzed

by

tyrosinase

immobilized

in

poly(ethyleneoxide)

conducting

polymers

Huseyin

Bekir

Yildiz

a,∗

_,

_Salim

_Caliskan

b

_,

_Musa

_Kamaci

a

_,

_Abdullah

_Caliskan

c

_,

Hasim

Yilmaz

c

a_Department_of_Chemistry,_Karamanoglu_Mehmetbey_University,₇₀₁₀₀_Karaman,_Turkey

b_Department_of_Mechanical_Engineering_and_Material_Science,_University_of_Pittsburgh,_PA_15261,_United_States c_Department_of_Chemistry,_Nevsehir_University,₅₀₃₀₀_Nevsehir,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15December2012

Receivedinrevisedform30January2013 Accepted30January2013 Available online xxx Keywords: Conductingpolymers Electropolymerization Biocatalysis Enzymeimmobilization l-Dopasynthesis

a

b

s

t

r

a

c

t

1-3,4-Dihydroxyphenylalaninecalledasl-Dopaisaprecursorofdopamineandanimportantneural messagetransmitterandithasbeenapreferreddrugforthetreatmentofParkinson’sdisease.Inthis study,withregardstothesynthesisofl-Dopatwotypesofbiosensorsweredesignedbyimmobilizing tyrosinaseonconductingpolymers:thiophenecappedpoly(ethyleneoxide)/polypyrrole(PEO-co-PPy) and3-methylthienylmethacrylate-co-p-vinylbenzyloxypoly(ethyleneoxide)/polypyrrole(CP-co-PPy). PEO-co-PPyandCP-co-PPyweresynthesizedelectrochemicallyandtyrosinaseimmobilizedby entrap-mentduringelectropolymerization.l-Tyrosinewasusedasthesubstrateforl-Dopasynthesis.Thekinetic parametersofthedesignedbiosensors,maximumreactionrateoftheenzyme(Vmax)andMichaelis

Mentenconstant(Km)weredetermined.Vmaxwerefoundas0.007␮mol/(minelectrode)forPEO-co-PPy

matrixand0.012␮mol/(minelectrode)forCP-co-PPymatrix.Km valuesweredeterminedas3.4and

9.2mMforPEO-co-PPyandCP-co-PPymatrices,respectively.OptimumtemperatureandpH,operational andshelflifestabilitiesofimmobilizedenzymewerealsoexamined.

1. Introduction

Enzymeswhicharewell-knowngreencatalysts,aresubstances that act ascatalysts in living organisms, regulating therate at which chemical reactions proceed without itself being altered in the process. They possess a high degree of specificity. The specificityinvolvesdiscriminationbetweensubstrates(substrate specificity),similarpartsofmolecules(regiospecificity),and opti-calisomers(stereospecificity)[1–6].Themildnessandspecificity ofenzymesendowthem witha highefficiencyforapplications in fine-chemical/pharmaceutical synthesis, food processing, biosensorsfabrication,bioremediation, and proteindigestion in proteomicanalysis[7–11].However,theapplicationsofenzymes arelimited bytheirinstabilityandnonreusability [12].Enzyme immobilizationisoneoftheeffectivewaystoovercomethese lim-itations;thetermimmobilizedenzymehasbeenusedtodescribe an enzyme that has been chemically or physically attachedto a water-insoluble matrix, polymerized into a gel or entrapped withina gelmatrix or microcapsule [13,14]. Immobilization of enzymehasseveraladvantages.Theseare:(i)thestabilityofthe enzymebyprotectingtheactivematerialfromdeactivation;(ii)

∗ Correspondingauthor.Tel.:+903382262000;fax:+903382262116. E-mailaddress:yildizhb@kmu.edu.tr(H.B.Yildiz).

repeateduse;(iii)signiﬁcantreductionintheoperationcost;(iv) easyseparationandrecoveryoftheenzyme[15].

Althoughthirtyyearshavepassedsinceconductingpolymers werediscovered,thescientistshavebeenstillfocusingondoing researchaboutthembecauseoftheirenormouspotentialfor appli-cation[16].Thesenewmaterials,alsocalledsyntheticmetals,can reachhighelectricalconductivity,veryclosetothevalueofsome metals and offeran extensivepossibility tomodify thesurface ofconventionalelectrodessupplyingfascinatingproperties[17]. Itwasfoundthattheycanbeusedinmanyareas,suchas elec-trochromicmaterials[18],organic-basedsolarcells[19,20],organic ﬁeldeffect transistors[21,22]and organiclight-emittingdiodes

[23,24].Moreover,providingsimplicityandhighreproducibilityin preparation,easinessinarrangingthethicknessoftheﬁlm, compat-ibilitywithbiologicalmolecules,andpossibilitytoproduceatroom temperaturemakeelectrochemicallysynthesizedCPscharmingin designingbiosensors[25,26].

Naturallyoccurringaminoacid1-3,4-dihydroxyphenylalanine (l-Dopa)isaprecursorofdopamineandanimportantneural mes-sagetransmitter[27].Ithasbeenapreferreddrugforthetreatment of Parkinson’s diseasesince 1967 [28,29]. Decrease in concen-trationof dopamineinthesubstantia nigraofthebrain causes Parkinsondisease [30,31]. Dopamine cannot beused asa drug forParkinsondiseasesinceitisunabletocrosstheblood–brain barrierwhereas l-Dopacan [32].l-Dopa canbeproducedfrom

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Scheme1. Routeforreactionforproductionofl-Dopa[40].

l-tyrosine by the help of the enzyme, tyrosinase (Scheme 1). Therefore, the analysis of tyrosinase activity is important for detectingmelanoma cells and Parkinson disease[33]. Polyphe-noloxidaseortyrosinase (E.C.1.14.18.1)is a coppercontaining enzyme which is one of themost versatile enzymesin nature

[34].Itiscommonlyfoundinmushrooms,yeast,bananas,grapes, apples,potatoes,frogs,andmammals[35].Moreover,tyrosinase haswidespreadapplicationsinindustry:electrochemicalbiosensor fordopamine[36],constructionofsensorstodeterminethe phe-nolicamountinwastewater[37],detectionofcatecholsinurine

[38],andobtainingtheconcentrationoftotalphenolicsinredwine

[39,40].

Inthisstudy,immobilizationoftyrosinasewasperformedvia entrapmentinconductingpolymersduringelectrochemical poly-merizationofpyrrolethroughthethiophenemoietyofthe poly-merswhichareCPandPEO(Schemes2and3).CP(3-methylthienyl methacrylate and p-vinylbenzyloxy poly(ethyleneoxide)) and PEO (thiophene capped poly(ethyleneoxide)) were synthesized and characterized in previous studies [41,42]. The conducting copolymers,CP-co-PPyandPEO-co-PPyweresynthesized electro-chemicallyusingsodiumdodecylsulfate(SDS)asthesupporting electrolyte.Thepurposeofthethisstudyistosynthesizel-Dopa (1-3,4-dihydroxyphenylalanine)whichisaprecursorofdopamine byusingimmobilizationoftyrosinaseinpoly(ethyleneoxide)type conducting copolymer matrices. By following this purpose and usingsubstrate,namelyl-tyrosine,optimumconditionsfor immo-bilizedtyrosinasesuchaspH,temperatureandkineticparameters (KmandVmax)wereinvestigated.Theoperationalandstorage

sta-bilitystudiesoftheseenzymeelectrodeswerealsostudied.

2. Materialandmethods

2.1. Material

Tyrosinase (E.C. 1.14.18.1), l-tyrosine, l-ascorbic acid, hydrochloric acid, sodium hydroxide, sodium molybdate, and sodiumnitritewerepurchasedfromSigmaandusedasreceived without further puriﬁcation. Sodium dodecylsulfate (SDS) was suppliedfromMerck.Pyrrole(Py)and3,4-ethylenedioxythiophene (EDOT) werepurchased fromAldrichand used withoutfurther puriﬁcation. NaH2PO4·H2O (sodium phosphate monobasic) and

NaHPO4·7H2O(sodiumphosphatedibasic)werepurchasedfrom

FisherScientiﬁcCompany.

2.2. Instrumentation

Potentioscan Wenking POS-73 and ST-88 potentiostats, Shi-madzu UV-160-A model spectrophotometer and Memmert D-91126modelwaterbathwereused.

2.3. Method

2.3.1. ImmobilizationoftyrosinaseinCP-co-PPyandPEO-co-PPy matrices

Immobilizationoftyrosinasewasperformedbyconstant poten-tialelectrolysisatroomtemperatureinatypicalthree-electrode cellcontainingCPcoatedplatinumfoil(1cm2₎_as_working,_bare_Pt

ascounterandAg/Ag+_as_reference_electrodes._Electrolysis_solution

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36 H.B.Yildizetal./InternationalJournalofBiologicalMacromolecules56 (2013) 34–40

C

O

CH

2

S

m

Scheme2.StructureofCP-co-PPyconductingcopolymer[41,50].

(1.2mg/mL),pyrrole(0.01M)and50mMphosphatebuffer(10mL, pH7.0).Polymerizationreactionswerecarriedoutbyapplying1.0V for40min.Afterelectrolysis,enzymeelectrodeswerewashedwith distilledwater inordertoremove bothexcesssupporting elec-trolyteandunboundenzymeandkeptinphosphatebufferat4◦C whennotinuse.

For the immobilization of tyrosinase in PEO-co-PPy matrix, asolutionof1mg/mLPPO,2mg/mL PEO,1.2mg/mLsupporting

S

CH

2

CH

2

OH

N

H

S

_N

N

O

H

n

CH

2

CH

2

n H

n

Scheme3. StructureofPEO-co-PPyconductingcopolymer[42].

electrolyte (SDS), 0.01M pyrrole and 10mL 50mM phosphate buffer(pH 7.0)was putin a typicalthree electrode cell. Three electrode cellhasthePtworkingand counter electrodesand a Ag/Ag+ _(0.01_M)_reference_electrode._{Immobilization}_was_carried

outataconstantpotentialof1.0Vfor40minatroomtemperature. Enzymeelectrodeswerekeptat4◦Cin50mMphosphatebuffer solution(pH7.0)whennotinuse(Scheme4).

2.3.2. Determinationoftyrosinaseactivity

Allexperimentsweredoneinconstanttemperaturewaterbath whileshaking. Theactivitiesofbiosensorsweredeterminedfor bothfreeandimmobilizedenzyme.Forfreeenzymeactivity,0.01M tyrosinasesolutionwasaddedtol-tyrosinesolutions(0.5–2.5mM) containingl-ascorbicacid.After30minreactiontime,enzymatic assaywasperformedbyadding1mlHCl(2M),1mlNaOH(2M) and1ml15%NaMo4andNaNO2solutionsinspeciﬁcreactiontimes

(5,10and 15min).For determiningtheimmobilizedtyrosinase activity,differentconcentrationsofl-tyrosineandl-ascorbicacid solutionswereprepared.Electrodeswereputintotesttubes con-tainingsubstratesolutions.Togetthedesirableconcentrationof l-Dopa,50minwasrequiredforCP-co-PPymatrix.Then,the enzy-maticassaymentionedabovewasperformed.Sinceformationof l-Dopacomplexistimedependent,l-Dopaconcentrationswere determinedbyspectrochemicalanalysisat460nmexactlyafter1h. Enzymeelectrodeswerekeptinphosphatebufferat4◦Cwhennot inuseanddailypreparedelectrodeswereusedinallexperimental steps[33].

2.3.3. DeterminationofoptimumpHandoptimumtemperature Forthetyrosinaseexperiments,theeffectofpHwasdetermined bychangingreactionmediumpHbetween3and11atconstant5Km

l-tyrosineconcentrationforbothsolubleandimmobilizedenzyme matrices.Theeffectoftemperaturewasdeterminedbychanging thereactionmediumtemperaturebetween10and80◦Cat con-stant5Kml-tyrosineconcentration.Inallexperimentstheenzyme

activitydeterminationexperimentswereperformedasdescribed in Section2.3.2 and relative enzymeactivitywascalculated by assigningthemaximumvalueofactivityas100%.

2.3.4. Operationalandstoragestabilityexperiments

Theoperationalstabilityofelectrodeswasstudiedby perform-ing40repetitivemeasurementsat25◦Cinapproximatelythree days.Storagestabilityof enzymeelectrodeswasdeterminedby checkingtheactivitieseverydayforaweekandthenoncein5days throughout40days.Intheinvestigationsofbothoperationaland storagestabilitiesactivitydeterminationprocedureswereusedin thesamewayasinSection2.3.2.Substrateconcentrationswere keptat5Kmandelectrodeswerestoredinbuffersolutionat4◦C

whennotinuse. AsdoneindeterminationofoptimumpHand temperatureexperiments,relativeenzymeactivitywascalculated byassigningthemaximumvaluesofactivityas100%inoperational andstoragestabilityexperiments.

2.3.5. Proteindetermination

Proteindeterminationmeasurementswereperformedby Brad-ford’smethod[43].Duringmeasurements,asolutionofBradford 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

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Scheme4. Schematicrepresentationofimmobilizationoftyrosinaseinconductingcopolymermatrices.

amountofproteinentrappedintheenzymeelectrodeduringthe electrolysis[39].

3. Resultsanddiscussion

3.1. Kineticparametersofenzymeelectrodes

Kineticstudiesoftheimmobilizedtyrosinasewereperformed atconstanttemperatureandpHwhilevaryingthesubstrate con-centration. Kinetic parameters include the maximum reaction rate(Vmax)oftheenzymaticreactionandtheMichaelis–Menten

constant(Km).Anenzymaticreactionreachesamaximum

veloc-ity (Vmax) when the substrate concentration is increased to

a level where there is a constant rate of product formation. The Michaelis–Menten constant (Km) deﬁnes the afﬁnity of

enzymetowarditssubstrate.LowertheKm valuemeanshigher

its afﬁnity against its substrate. Maximum velocity (Vmax) and

Michaelis–Menten constants (Km) for enzyme electrodes were

foundfromaLineweaver–Burkplot[44]whichisaplotof1/V0

against1/[S0]for systems obeyingtheMichaelis–Menten

equa-tion. The graph being linear can be extrapolated at anywhere approximatingtoasaturatingsubstrateconcentration,evenifno experimenthasbeenperformedandfromtheextrapolatedgraph, thevaluesofKmandVmaxcanbedetermined.

Intheimmobilizationoftyrosinaseforl-Dopasynthesis exper-iments,theKmvalueforthefreetyrosinasewasfoundas6.4mM

l-tyrosine.KmofPEO-co-PPy/tyrosinaseelectrodesdecreased

com-pared to that of the free enzyme. The substantial decrease in Kmvalueleadstothetendencyofenzymetobinditssubstrate

more strictly than the free enzyme and CP-co-PPy/tyrosinase electrode do, hence, enzyme substrate complex stays together for a long time that makes the enzymatic reaction rate of

PEO-co-PPy/tyrosinaseelectrodetheslowest.Besides,smallerKm

valuethanfreetyrosinaseindicatethatthePEO-co-PPymatrix pro-videsamicroenvironmentwhichismoresuitablethanthatinthe solution.ThehighestKmvaluewasobserveduponthe

immobiliza-tionofenzymeinCP-co-PPymatrix.Itisresultedfromthelower afﬁnityofenzymetowarditssubstrate,whichisprobablycaused bythestructuralchangeoftheenzymeuponentrapmentinthe matrix.Thedecreaseinthereactionrate,whichwasobservedfor immobilizedenzymeinCP-co-PPymatrixwithrespecttofreeone mightberesultedfromthedifﬁcultyindiffusionofsubstratetothe matrixascomparedtodiffusioninsolution.Kmvalueoftyrosinase

enzymeimmobilizedinCP-co-PPycopolymerwasfoundas9.2mM, andthatofPEO-co-PPymatrixas3.4mM.Theefﬁciencyfactor(₎ is theratio of themaximum reactionrates of theimmobilized enzymeoverthatofthefreeenzyme.Valuesofwerecalculated fromthisratioandwerefoundas0.39forPEO-co-PPyand0.67for CP-co-PPymatrices.Vmax/Kmisthecatalyticefﬁciency(kb)ofan

enzyme–substratepair.Catalyticefﬁciencyofthefreetyrosinase wascalculatedas0.0028.Itwasfoundas0.0021and0.0013for PEO-co-PPyandCP-co-PPymatrices,respectively(Table1).

3.2. Proteindeterminationforenzymeelectrodes

Results of protein determination experiment for PEO-co-PPy/tyrosinase and CP-co-PPy/tyrosinase electrodes were 3.27×10−3 and 3.32×10−3mg protein, respectively. Although tyrosinaseenzyme entrappedin CP-co-PPyelectrodeis approx-imately thesame asthe enzymeholdin PEO-co-PPy electrode, maximum reaction rate observed for tyrosinase entrapped in CP-co-PPy is 1.7 times faster than the rate observed for PEO-co-PPy. This indicates that activity of an immobilized enzyme is dependent not only on the entrapped enzyme quantity but

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Table1

Kineticparametersoffreeandimmobilizedtyrosinaseenzymeforl-Dopasynthesis.

Vmax(␮mol/(minelectrode)) Km(mM) Efﬁciencyfactor, Catalyticefﬁciency,kb

Freetyrosinase 0.018a _6.4 _– _0.0028

PEO-co-PPy/tyrosinase 0.007 3.4 0.39 0.0021

CP-co-PPy/tyrosinase 0.012 9.2 0.67 0.0013

a_␮mol_min−1_mL−1_.

alsointeractionbetweenenzymeandmatrix.Thus,theeffective activity of enzyme electrode might be lower than the activity expectedfromthequantityentrappedasaresultofdeactivation uponimmobilization.

3.3. Effectoftemperature

Theeffectoftemperaturebetween10and80◦Conthe rela-tiveenzymeactivitywasinvestigatedandillustratedinFig.1.In apreviousstudymaximumactivityoffreetyrosinaseshoweda maximumactivityatabout30◦C[40].However,maximumenzyme activitiesforCP-co-PPyandPEO-co-PPymatriceswereat50and 60◦Crespectively.Afterthesetemperaturesupto80◦C,although PEO-co-PPy/tyrosinaseelectrode lost35%ofitsenzymeactivity, CP-co-PPy/tyrosinaseelectrodealmostallitsenzymeactivity.The PEO-co-PPymatrixshowedhighstabilitybetween30and70◦C. 3.4. EffectofpH

Foroptimization ofpHat 25◦C,the pHofthe mediumwas changedbetween3and11.Duetothedenaturationoftyrosinase inacidicmedialowerthanpH3,thepHoptimizationstudystarted frompH3forenzymeelectrodes.Forfreeenzyme[40],and CP-co-PPymatrixmaximumactivitywasobservedaroundpH7.Although thePEO-co-PPyenzymeelectrodeshowsaslightmaximumatpH 8ithasenzymeactivityhigherthan80%inthepHrangeof6–10. Therefore,immobilizationofenzymeinthecopolymermatrix sup-pliesnotonlyahigherstabilityagainstpHbutalsoaworkingrange betweenpH6and10(Fig.2).Besides,bothmatriceshavesuitable environmentforenzymeandbothbiosensorshavetheadvantages inmedicalapplicationssincethebloodpHis7.4.Fortheenzyme electrodes,maximumactivitywasshiftedtowardthealkalineside whencompared tothat ofthefreeenzyme. Thisalkaline range stabilityoftheimmobilizedenzyme,whichisobservedfor tyrosi-nasecanbeexplainedbythebufferingeffectofthedopantion.The dopantionusedintheelectropolymerizationpenetratesintothe matrixasthepolymerisoxidized.Resultingconductingpolymer

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

R

el

ati

ve

E

nzy

m

e A

cti

vi

ty

Temperature

(°C)

Fig.1. Effectofincubationtemperatureonactivityoftyrosinaseimmobilizedin CP-co-PPy()andPEO-co-PPy()matrices.

haspositivechargesonitsownandcanbeassumedthatithasa negativechargeshellaroundit.Thisnegativechargeshellattracts H+_ions_around_itself._Those_H+_ions_protect_the_enzyme

encapsu-latedinthematrixfromthehighconcentrationofOH−ionsand giveanextrastabilitytotheenzymeinalkalineside[45].Itcanbe seenkineticparameters,optimumpHandtemperaturevaluesof differenttyrosinaseimmobilizationmethodsforl-Dopasynthesis inTable2.

3.5. Operationalandstoragestabilityofenzymeelectrodes

Enzymescaneasilylosetheircatalyticactivityanddenatured. Thereforeoperationalandstoragestabilityareimportant consider-ationsforanimmobilizedenzyme.Operationalstabilityofenzyme electrodeswastriedtoestimatethestabilityofelectrodesinterms of40repetitiveuses;PEO-co-PPy/tyrosinaseelectrodemaintained anactivityat80%untiltheassaynumber15andexhibitedagood stabilityupontherepetitiveuses.After15thassay,enzymeactivity decreasedandafter33rdassayitstayedconstantat55%ofits origi-nalactivity.Inspiteofhavinggoodenzymeprotectionagainsthigh temperatureandpH,PEO-co-PPymatrixdidnotprotectenzyme verywell.CP-co-PPy/tyrosinaseelectrode showeda high opera-tionalstabilityandretained95%ofitsoriginalactivityuntilthe assaynumber15andthenkept85%ofitsactivityevenafter40thuse (Fig.3).TheslightincreaseintheresponseofCP-co-PPy/tyrosinase electrodeisrelatedtotheswellingofthepolymerstructureand reorganizationoftheenzymemoleculesinthismatrix[46].

Storage stability of tyrosinase immobilized in PEO-co-PPy exhibitsan50%lossofitsactivityin25daysandstayedconstant untilendoftheitsstoragestabilityexperiment.Ontheotherhand, tyrosinaseenzymeimmobilizedinCP-co-PPymatrixlost15%ofits activityintheﬁrst15daysandthenstayedconstantwith80%of itsoriginalactivityuntil40thday.Bothelectrodeshaveverygood stabilitiesintheﬁrst5daysandcanbesafelyusedinthisperiod. SinceimmobilizedtyrosinaseinCP-co-PPymaintained80%ofits originalactivityafter15thdayandstayedconstantupto40thday,

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

R

el

ati

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E

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pH

Fig.2.EffectofpHonactivityoftyrosinaseimmobilizedinCP-co-PPy()and PEO-co-PPy()matrices.

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Table2

Differenttyrosinaseimmobilizationmethodsforl-Dopasynthesis.

Matrices Vmax Km(mM) pH Temperature(◦C) References

PPy 0.013a _3.7 _7.0 ₃₀ _[40]

PEDOT 0.040a _5.2 _7.0 ₃₀ _[40]

P(SNSNO2)-co-PPy 0.020a 2 7.0 60 [47]

Magneticbeads 1.05b _1.04 _6.0 ₄₀ _[33]

Carbonnanoparticle-PPy 0.0036 0.21 6.5 Ambient [48]

Cross-linkedtyrosinaseaggregates – – 5.5 30 [49]

a_␮mol/(min_electrode). b_␮mol/(min_mg_protein). 0 10 20 30 40 50 0 20 40 60 80 100 120

Rel

at

ive Enz

yme Act

ivi

ty

Assay Number

Fig. 3. Operational stabilities of CP-co-PPy/tyrosinase () and PEO-co-PPy/tyrosinase()electrodes. 0 10 20 30 40 50 0 20 40 60 80 100 120

R

el

ati

ve

E

nzy

m

e A

cti

vi

ty

Days

Fig.4.StoragestabilityofCP-co-PPy/tyrosinase()andPEO-co-PPy/tyrosinase() electrodes.

itcanalsobeusedbetweenthe15thand40thdayswithahigh activity(Fig.4).

4. Conclusion

Production of l-Dopa was achieved using tyrosinase immo-bilized on conducting polymers CP-co-PPy and PEO-co-PPy. Kineticparameters,operationalandstoragestabilities,optimum temperatureandpHwereinvestigatedforthematrices. PEO-co-PPy/tyrosinase electrode had the smallest Km and Vmax values

when compared with Km and Vmax values of free enzyme and

CP-co-PPy/tyrosinaseelectrode.ThesmallestKmvalueshowsthat

PEO-co-PPymatrixprovidesamicroenvironmentwhichismore suitablethanthatinthesolutionandCP-co-PPymatrix.Itcanbe

understoodthatenzymeimmobilizedinPEO-co-PPymatrixbinded itssubstratemorestrictlythanthefreeenzymeinthesolutionand enzymeimmobilizedinCP-co-PPymatrixdid.Inspiteofhaving goodenzymeprotectionagainsthightemperatureandpH, opera-tionalandstoragestabilitiesofPEO-co-PPywerepoorbecauseof notprotectingenzymeverywell.Thisstudyprovesthatconducting polymers;CP-co-PPyandPEO-co-PPycanbeusedas immobiliza-tionmatricesfortyrosinaseintheproductionofl-Dopa.

Acknowledgements

AuthorswouldliketothanktheScientificandTechnological Research Council of Turkey (TUBITAK Grant Number 109T439) andtheScientific ResearchProjectsFoundationofKaramanoglu MehmetbeyUniversity(KMU-BAPGrantNumber09-M-11)forthe financialsupportofthisresearch.

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