laccase-catalyzed synthesis of conducting polyaniline

Enzyme and Microbial Technology 33 (2003) 556–564

Laccase-catalyzed synthesis of conducting polyaniline

Alexey V. Karamysheva, Sergey V. Shleevb, Olga V. Korolevab,Alexander I. Yaropolovb, Ivan Yu. Sakharova,c,∗

a Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119992, Russiab Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow 119071, Russia

c Division of Chemistry, G.V. Plekhanov Russian Economic Academy, Moscow 113054, Russia

Received 4 October 2002; received in revised form 30 April 2003; accepted 3 May 2003

Abstract

Laccase isolated fromCoriolus hirsutus was first used in the synthesis of water-soluble conducting polyaniline. The laccase-catalyzedpolymerization of aniline was performed in the presence of sulfonated polystyrene (SPS) as a template. Laccase shows remarkableadvantages in the synthesis of conducting polyaniline compared to the commonly used horseradish peroxidase due to its high activityand stability under acidic conditions. The characterization of the polyelectrolyte complex of polyaniline and SPS has been carried outusing UV-Vis and FTIR spectroscopy. Cyclic voltammetry and dc conductivity measurements confirmed that electroactive polyaniline wassynthesized by the laccase-catalyzed polymerization of aniline.© 2003 Elsevier Inc. All rights reserved.

Keywords: Laccase;Coriolus hirsutus; Polyaniline

1. Introduction

In recent years there is a tremendous interest in the pro-duction of conducting polymers. Polyaniline is one of themost important conducting polymers, which may be usedas active component of organic lightweight batteries, micro-electronics, optical display, for anticorrosive protection, inbioanalysis, etc.[1–3], due to its good electrical and opticalproperties as well as high environmental stability. Polyani-line is commonly synthesized by oxidizing aniline monomerunder strongly acidic conditions (usually in 1 M H2SO4 or1N HCl) at∼0◦C using ammonium persulfate as an initiatorof oxidative polymerization[4,5]. In the course of the syn-thesis the emeraldine base of polyaniline is formed (Eq. (1)).The emeraldine base of polyaniline is not a conductor; onlyits derivative, emeraldine salt is conductive form. Emeral-dine salt is usually obtained from emeraldine base via proto-nation of its imine sites with sufficiently strong acids such asorganic sulfonic and phosphoric acids and their derivatives(Eqs. (2) and (3))[6]. This process is named “doping”.

Several drawbacks of this method seriously limited its ap-plication for production of conducting polyaniline. First, thereaction is a polymerization process, which is not kineticallycontrollable. Second, the reaction conditions are not environ-

∗ Corresponding author. Tel.:+7-095-9393407; fax:+7-095-9392742.E-mail address: [email protected] (I.Yu. Sakharov).

+ 2 A-

+ 2 H+

HN

HN

HN

HN

A- A-

..

HN N N

HN

acid-doped PANI

emeraldine base

+ +[

[ ]

]

n

n

HN N N

HN[ ]

n

(1)

(2)

(3)

H

+

H

+A-

A-

mental friendly due to the use of high concentration of strongacids in the reaction system. Third, the synthesized conduct-ing polyaniline is not processable due to the poor solubilityin common solvents. This is due to intermolecular hydrogenbonding which is formed between amine groups and iminegroups of the adjacent chains acting as acceptors[1].

Efforts have been made to increase solubility of con-ducting polyaniline. These approaches involved the modi-fication of either the benzene rings or the N–H groups of

0141-0229/$ – see front matter © 2003 Elsevier Inc. All rights reserved.doi:10.1016/S0141-0229(03)00163-7

A.V. Karamyshev et al. / Enzyme and Microbial Technology 33 (2003) 556–564 557

polyaniline repeat units with different functional groups(–CH3, –OCH3, –SO3, long alkyl chain)[7–9]. Such modi-fications led to the improvement of the solubility in commonsolvents. For example, the treatment of polyaniline withfuming sulfuric acid allowed introducing a sulfonic groupon the benzene ring of polyaniline repeat unit[8,10]. Theresulted sulfonated polyaniline is self-doped and solublein water. Unfortunately, many of synthesized sulfonatedpolyanilines are water-soluble only at higher pHs where thepolymer is in its undoped form[11].

An alternative approach of the synthesis of water-solubleconducting polyaniline has been recently developed by in-troducing negatively charged polyelectrolyte into the reac-tion system[12–14]. In this case, the polyelectrolytes willemulsify the aniline monomer prior to the polymerization,and help to dope and dissolve the synthesized polymer.Most importantly, the polyelectrolyte molecules such as sul-fonated polystyrene (SPS) will serve as template to pro-mote a head-to-tail coupling during polymerization of ani-line, which is necessary for the formation of conductingpolyaniline[15].

With hazardous waste becoming increasingly expensive totreat, biochemical reactions are more attractive as alternativeroutes for synthesis of fine chemicals[16–18]. This way wasalso used in polymer chemistry[19]. Because horseradishperoxidase oxidizes aniline[15,20], this enzyme has beenused in the synthesis of water-soluble polyaniline. The re-action was carried out in the presence of hydrogen peroxideas a reducing substrate and some polymeric templates hav-ing sulfonic and phosphoric groups. The advantage of theenzymatic approach compared to chemical one is that thesynthesis has been carried out under mild conditions with-out generating toxic by-products.

Unfortunately, horseradish peroxidase shows the low ac-tivity and stability at pH below 4.5[15,21,22], i.e. in the pHinterval where the polyelectrolyte complex between polyani-line (pKa of aniline is 4.63) and charged polymeric templatesis formed. This results in a consumption of large amountof the peroxidase in the polymerization. For developmentof more technological processes alternative oxidoreductasescapable effectively to polymerize aniline under acidic con-ditions should be used.

To address this issue, a new biocatalyst, laccase, which cancatalyze the oxidation of aniline in the presence of molecu-lar dioxygen (O2), is introduced in synthesis of conductingpolyaniline. Laccase (EC 1.10.3.2) is one of the oxygen oxi-doreductases which is capable of oxidizing a lot of inorganicand organic substrates including aniline and its derivatives[23]. In our previous works, we have purified some laccasesfrom Coriolus hirsutus, Coriolus zonatus andCerena max-ima [24–26]. Preliminary results have shown that the lac-caseC. hirsutus is active and stable under acidic conditions[25]. In this paper, we report a detailed investigation onthe synthesis of water-soluble conducting SPS–polyanilinecomplex using laccase isolated from culture medium ofC.hirsutus as a catalyst.

2. Experimental section

2.1. Materials

Aniline, Na2HPO4 and citric acid were purchased fromSigma Chemical Co. (St. Louis, MO, USA). Aniline waspurified by distillation before use. SPS (MW 70,000) wasobtained from Aldrich (Milwaukee, WI) and used as avail-able.

Laccase was isolated from the culture fluid ofC. hirsutus.The procedure for the purification was described elsewhere[25]. The specific activity of laccase measured toward cate-chol was not less than 350 units mg−1 of protein.

2.2. Laccase activity

The activity of laccaseC. hirsutus was usually determinedspectrophotometrically in 100 mM acetate buffer (pH 4.2),containing 10 mM catechol as substrate, and the absorbancechange at 410 nm was measured at 25◦C for 60 s. The valueof extinction coefficient for the oxidized products of catecholis 740 M−1 cm−1.

One unit of activity is defined as the amount of laccaseoxidizing 1�mol of substrate per min under standard con-ditions. Specific activity is expressed as units of activity permg of protein.

2.3. Enzymatic polymerization of aniline

The synthesis of SPS–polyaniline complexes was carriedout usually in 0.1 M citrate–phosphate buffer, pH 3.5–4.4with or without stirring at ambient temperature in humiditychamber. The concentration of monomer aniline and SPSin feed varied from 5 to 125 mM. The concentration of lac-case in feed was usually 5.5 × 10−7 M. The reaction oflaccase-catalyzed polymerization of aniline was evaluatedusing UV-Vis spectroscopy.

2.4. Spectroscopic methods

The electronic spectra were recorded on a ShimadzuUV-2401 PC spectrophotometer. In each measurement dis-tilled water was used as a control. The FTIR spectra wererecorded on a spectrophotometer Nicolet Magna-750 usingKBr pellets.

2.5. Cyclic voltammetry

Electroanalytical experiments of polyaniline sampleswere performed using a BAS CV-50W Voltammetric Anal-yser (Bioanalytical System, USA). SPS–polyaniline wasdissolved in a solution containing 0.01 M HCl and 0.1 MNaCl. The solution was loaded in a one-compartment elec-trochemical cell (volume 10 ml) consisted of an Ag/AgClreference electrode, a platinum wire counter electrode and

558 A.V. Karamyshev et al. / Enzyme and Microbial Technology 33 (2003) 556–564

a 1 mm diameter platinum working electrode. The potentialscan range was−100 to 1200 mV. Cyclic voltammogramswere recorded with a scan rate of 100 mV s−1.

2.6. Conductivity

The electric conductivity of SPS–polyaniline was mea-sured with a four-probe instrument. The sample was firstdialyzed against 1 mM HCl and then freeze-dried. A roundpellet was produced from the dried sample for the measure-ment of conductivity.

3. Results and discussion

3.1. Enzymatic polymerization

Typically, peroxidase-catalyzed polymerization of con-ducting polyaniline is carried out at pH∼ 4.0, which is notthe optimal pH for HRP catalysis. Under these conditionsHRP usually loses its catalytic activity a very short time pe-riod, which results in consumption of large amount of theenzyme during the polymerization reaction. Since laccaseC. hirsutus is active and stable under acidic conditions[25],it will have a great advantage in comparison to HRP in thetemplate-assisted polymerization of aniline.

The laccase-catalyzed polymerization of aniline was stud-ied in pH range from 3.5 to 4.4. The reaction was carriedout at ambient temperature in the presence of SPS as men-tioned in previous works[15]. The reactions were monitoredwith UV-Vis absorption spectroscopy. As shown inFig. 1,

wavelength, nm

300 400 500 600 700 800 900

abso

rban

ce

0.0

0.5

1.0

1.5

pH 3.7pH 3.9

pH 4.2pH 4.4pH 3.5

Fig. 1. Effect of pH on the rate of aniline polymerization in the presence of SPS catalyzed by laccase at 20◦C. The experimental conditions: 0.1 Mcitrate–phosphate buffer; [aniline]= [SPS]= 6 mM; [laccase]= 5.5 × 10−7 M; the spectra were recorded 15 min after the polymerization initiation.

strong polaron absorption band at around 700–780 nm canbe observed at pH 3.5, 3.7 and 3.9 indicating on the forma-tion of conducting polyaniline. Therefore, we can concludethat laccase can catalyze the polymerization of aniline in thepresence of SPS to form water-soluble conducting polyani-line under mild conditions compared to traditional chemicalmethod[4,5].

Since the presence of O2 is necessary for laccase catalyticaction, agitation should strongly affect the progress of reac-tion. This speculation was confirmed by comparative study-ing of the enzymatic polymerization with and without stir-ring (Fig. 2A). The reaction under constant stirring showsa higher reaction rate. The agitation appears to change therate not only due to an increase of mass transfer of react-ing compounds but maintenance of constant concentrationof O2 consuming in the course of the polymerization.

The stability of laccase was also investigated under theconditions of aniline polymerization. In this study, we alsouse the absorbance at 760 nm as an indicator of polyanilineproduction. Laccase was active in the solution for 4–5 days,demonstrating its high operational stability under the reac-tion conditions (Fig. 2B).

3.1.1. Effect of concentration of aniline and SPS on anilinepolymerization

The increase of aniline concentration up to 75 mM infeed results in the increase of the polymerization rate(Fig. 3). However, further increasing of aniline concentra-tions led to laccase inhibition by the substrate. Because offorming viscous final product solution in feed at 75 mManiline concentration, preparative synthesis of polyelec-


Fig. 2. Kinetics of the polymerization reaction of aniline in the presence of SPS and laccase with (circles) and without (triangles) stirring. The experimentalconditions: 0.1 M citrate–phosphate buffer, pH 3.5; [aniline]= [SPS]= 50 mM; [laccase]= 5.5 × 10−7 M.

trolyte SPS–polyaniline complexes was usually carried outat 50 mM aniline.

The effect of SPS concentration on the polymerizationwas also evaluated. It was shown that the increase of SPSconcentration in feed results in the decrease of the rate of theaniline polymerization (Fig. 4). We have speculated that itwas due to a change of oxygen concentration in the reactionmedium in the presence of the template polymer. However,the measurement of oxygen concentration using a Clark’selectrode showed no difference in solutions with different

[aniline], mM

0 25 50 75 100 125 150

abso

rban

ce a

t 760

nm

25

50

75

100

Fig. 3. Effect of concentrations of aniline on rate of the template production of water-soluble SPS–polyaniline complexes by laccase. The experimentalconditions: 0.1 M citrate–phosphate buffer, pH 3.7; [SPS]= 6 mM; [laccase]= 5.5 × 10−7 M; the spectra were recorded 5 days after the polymerizationinitiation.

SPS concentration. Thus, the presence of SPS does not affectthe oxygen concentration in the solution.

The decrease of laccase activity with increasing of SPSconcentration can also be considered as another possible rea-son for the change of reaction rate. To evaluate it, the effect ofSPS concentration on the laccase activity measured againstcatechol, a specific substrate for laccase, was determined.However, the rate of catechol oxidation did not depend onthe SPS concentration under experimental conditions usedand, therefore, SPS does not affect the laccase activity.


Fig. 4. Effect of concentrations of SPS on rate of the template production of water-soluble SPS–polyaniline complexes by laccase. The experimentalconditions: 0.1 M citrate–phosphate buffer, pH 3.7; [aniline]= 50 mM; [laccase]= 5.5×10−7 M; the spectra were recorded 2 days after the polymerizationinitiation.

Comparison of chemical structures of aniline and catecholshowed that in contrary of the latter compound aniline car-ries a positive charged group (–NH3

+) and, therefore, mayform ionic complexes with SPS under experimental condi-tions. At increasing SPS concentration an equilibrium be-tween aniline, SPS and aniline–SPS complex is shifted to-ward the formation of the complex leading to removal ofaniline from reaction mixture. Thus, we suggest that the de-crease of aniline polymerization rate is connected with de-crease of free aniline concentration in solution due to for-mation of aniline–SPS complexes.

This conclusion allows us to suppose a mechanism of thetemplate synthesis of polyaniline in the presence of laccase.At mixing of the reacting compounds a part of aniline mole-cules reacts with SPS forming their complexes, whereas theother part stays in a free state. After addition of the en-zyme free aniline molecules are oxidized by the laccaseand their radical products produced during the enzymaticstep migrate to SPS–aniline complexes. There these radicalsinitiate template polymerization of aniline with formationof SPS–polyaniline complex. We suggest that some radi-cals formed may react with free aniline molecules in bulk.However, possibility of the latter reaction is much lower,as aniline concentration in its complex with SPS should besignificantly higher than that in volume. Even if some partof aniline molecules reacts in bulk, the oligomeric prod-ucts formed produce immediately polyelectrolyte complexeswith SPS. The proposed mechanism has been confirmed byUV-Vis spectroscopy detecting only existence of polyanilinebound with SPS. Further alignment of polyaniline chain ap-pears to be carried out due to a reaction of aniline oligomers

stated in the complex with radical products formed by theenzyme.

The data obtained permitted to compare the laccase fromC. hirsutus and horseradish peroxidase as catalysts of ani-line polymerization. Although both enzymes catalyze effec-tively the oxidative polymerization of aniline, the laccaseshows higher operational stability under the reaction condi-tions. It should be emphasized also that in the contrary tothe peroxidase which loses its activity in the presence ofits oxidizing substrate (hydrogen peroxide)[27,28], the lac-case is not inactivated by oxygen (the oxidizing agent oflaccase). These features make the laccase more attractivefor production of conducing polyaniline than horseradishperoxidase.

3.1.2. Characterization of water-soluble SPS–polyanilinecomplexes

The solubility of the synthesized complex of SPS andpolyaniline is strongly depended on the feed ratio of SPS toaniline. One day after initiation of the aniline polymerizationall polyelectrolyte complexes obtained at different ratios ofmolar concentrations of repeating unit of SPS and anilinein feed (1:3, 3:5 and 1:1) were soluble in water. However,later for the two former complexes we detected the forma-tion of precipitates. Only the polyaniline sample obtainedat a ratio 1:1 of molar concentrations of repeating unit ofSPS and aniline in feed was soluble completely. Such poly-electrolyte complex was stable at 4 and 25◦C for 6 monthsas a minimum. This complex dialysed against 1 mM HCland freeze-dried may be re-dissolved in aqueous solutionsor N-methylpyrrolidone.


Fig. 5. Effect of pH on UV-Vis spectra of aqueous solution of the SPS–polyaniline complex. The pH values of the complex varied by its titration withNaOH and HCl solutions. SPS–polyaniline was produced enzymatically at pH 3.7 and a molar ratio of concentration of repeating unit of SPS and anilineof 1:1 ([aniline]= [SPS]= 50 mM).

3.2. Doping and dedoping

The SPS–polyaniline complex produced at the optimalconditions (pH 3.7 and a molar ratio of SPS and anilineof 1:1) has been characterized in detail. As seen inFig. 5,electronic spectrum of the SPS–polyaniline complex underacidic conditions exhibits three characteristic absorptionbands. The first absorption band at 320–360 nm arises from�–�∗ electron transition within benzenoid segments. Thesecond (400–420 nm) and third (760 nm) absorption bandsare related to doping level and formation of polaron ofthe conducting form, respectively[29,30]. The former twopeaks are combined into a single flat peak as describedpreviously[31].

The shift of the position of the third peak as a functionof pH of the reaction medium was observed at titration ofthe SPS–polyaniline complex by NaOH and HCl (Fig. 5).At decreasing pH from 3.7 to 2.9 the electronic spec-trum did not change, whereas the titration of the polyani-line complex using sodium alkali changed completely theUV-Vis spectrum. The increase of the pH value in thepolyaniline complex solution clove ionic bounds formedbetween the polymers, and the polyaniline become un-doped that is reflected in change of UV-Vis spectra (Fig.5). The transition point from doped to undoped form laysbetween pH 6.2 and 7.5. As pH values above the transi-tion point (under alkaline conditions), the bands at 420and 760 nm disappear, and a strong absorption at 556 nmbegins to emerge. Previously the appearance of band at550–580 nm was observed also for aqueous solutions ofpolyaniline-co-2-acrylamido-2-methyl-1-propanesulfonic

acid, polyelectrolyte complex of PANI and SPS and sul-fonic acid ring-substituted PANI under strongly alkalineconditions[15,32,33]. Here it should be noted that for PANIdissolved in organic solvents the same band is observed ataround 640 nm[1].

3.3. FTIR

The FTIR spectra of SPS and SPS–polyaniline com-plex are presented inFig. 6. Comparison of these spectrashowed the existence of some bands in the spectrum ofthe complex characteristic for polyaniline. The peaks at1560 and 1488 cm−1 are assigned to a C=C ring stretchingin quinoid and benzenoid units of polyaniline in dopedform [13,34,35]. The sulfonic groups of SPS show charac-teristic bands at 1180 and 1042 cm−1 [32]. However, theformation of some ionic bonds between sulfonic groupsof SPS and imine groups of polyaniline in the complexshifts this value to 1215 cm−1. The presence of the peak at1299 cm−1 characteristic for Caromatic–N stretching vibra-tion confirms that polyaniline in the complex is in dopedform, because for undoped polyaniline the same peak isrevealed at 1310–1313 cm−1 [32,36]. The peaks at 799 and880 cm−1 indicate head-to-tail coupling of the monomerwith the formation of linear polymeric chains[35].

3.4. Electroactivity

Aqueous solution of the SPS–polyaniline complex in0.01 M HCl, pH 2.0 was electrochemically active, as canbe seen from its cyclic voltammogram inFig. 7. For this


Fig. 6. FTIR spectra of (a) SPS and (b) SPS–polyaniline complex. SPS–polyaniline was produced enzymatically at pH 3.7 and a molar ratio ofconcentration of repeating unit of SPS and aniline of 1:1 ([aniline]= [SPS]= 50 mM).


Fig. 7. Cyclic voltammetry of the SPS–polyaniline complex solution in 0.01N HCl, pH 2.0. Voltammogram was recorded with a scan rate of100 mV s−1. SPS–polyaniline was produced enzymatically at pH 3.7 and a molar ratio of concentration of repeating unit of SPS and aniline of 1:1([aniline] = [SPS]= 50 mM).

complex two reduction peaks were observed in the ca-thodic sweep. The first cathodic peak is broad, not wellresolved and pseudoreversible asymmetric with anodicpeak observed in the potential range at 350–550 mV versusAg/AgCl. The second cathodic irreversible peak has strongcurrent maximum at potential about 50 mV. To compare theresults obtained with those from the literature it should benoted that cyclic voltammograms for polyaniline producedenzymatically by laccase and horseradish peroxidase[15]or electrochemically[37] were not identical that gives toresearchers broad possibilities for production of polyanilinewith different electrochemical characteristics.

The dc conductivity of the SPS–polyaniline complex wasmeasured with a four-probe method. The conductivity valueof the complex was 2× 10−4 S cm−1. Although this magni-tude is not high in comparison with best samples of dopedpolyaniline (approximately several hundreds S cm−1 [1]), itis similar to those measured previously for water-solublepolyanilines produced by other methods[15,35]. It shouldbe noted that in many cases, for polyanilines, being solu-ble and, hence, processable, is sometimes more important

than being highly conductive[10]. Thus, high solubility inaqueous solutions of the enzymatically obtained polyanilinecomplexes compensates their drawback connecting with itsnot enough high conductivity.

Acknowledgments

The authors thank Prof. Boris V. Lakshin for FTIR analy-sis, Dr. Elena V. Stepanova for the preparation of the enzymepreparation and Dr. Lynne Samuelson and Dr. Wei Lui forvaluable discussions and conductivity measurements. Thiswork was supported by the Russian Foundation of BasicResearches (Grant 02-04-48885).

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laccase-catalyzed synthesis of conducting polyaniline

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