Laccase-catalyzed synthesis of conducting polyaniline

Enzyme and Microbial Technology 33 (2003) 556–564

Laccase-catalyzed synthesis of conducting polyaniline Alexey V. Karamyshev a , Sergey V. Shleev b , Olga V. Koroleva b , Alexander I. Yaropolov b , Ivan Yu. Sakharov a,c,∗ a

Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119992, Russia b 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 from Coriolus hirsutus was first used in the synthesis of water-soluble conducting polyaniline. The laccase-catalyzed polymerization of aniline was performed in the presence of sulfonated polystyrene (SPS) as a template. Laccase shows remarkable advantages in the synthesis of conducting polyaniline compared to the commonly used horseradish peroxidase due to its high activity and stability under acidic conditions. The characterization of the polyelectrolyte complex of polyaniline and SPS has been carried out using UV-Vis and FTIR spectroscopy. Cyclic voltammetry and dc conductivity measurements confirmed that electroactive polyaniline was synthesized 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 production of conducting polymers. Polyaniline is one of the most important conducting polymers, which may be used as active component of organic lightweight batteries, microelectronics, optical display, for anticorrosive protection, in bioanalysis, etc. [1–3], due to its good electrical and optical properties as well as high environmental stability. Polyaniline is commonly synthesized by oxidizing aniline monomer under strongly acidic conditions (usually in 1 M H2 SO4 or 1N HCl) at ∼0 ◦ C using ammonium persulfate as an initiator of oxidative polymerization [4,5]. In the course of the synthesis the emeraldine base of polyaniline is formed (Eq. (1)). The emeraldine base of polyaniline is not a conductor; only its derivative, emeraldine salt is conductive form. Emeraldine salt is usually obtained from emeraldine base via protonation of its imine sites with sufficiently strong acids such as organic 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 application for production of conducting polyaniline. First, the reaction is a polymerization process, which is not kinetically controllable. 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).

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

[

H N

H N

N

N

]n (1)

emeraldine base + 2 A+ 2 H+

[

[

H N

H N +

. +H N A-

H N

H N

+

A-

A

H N

+H . NA

-

]n (2)

H N

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acid-doped PANI

mental friendly due to the use of high concentration of strong acids in the reaction system. Third, the synthesized conducting polyaniline is not processable due to the poor solubility in common solvents. This is due to intermolecular hydrogen bonding which is formed between amine groups and imine groups of the adjacent chains acting as acceptors [1]. Efforts have been made to increase solubility of conducting polyaniline. These approaches involved the modification of either the benzene rings or the N–H groups of

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

polyaniline repeat units with different functional groups (–CH3 , –OCH3 , –SO3 , long alkyl chain) [7–9]. Such modifications led to the improvement of the solubility in common solvents. For example, the treatment of polyaniline with fuming sulfuric acid allowed introducing a sulfonic group on the benzene ring of polyaniline repeat unit [8,10]. The resulted sulfonated polyaniline is self-doped and soluble in water. Unfortunately, many of synthesized sulfonated polyanilines are water-soluble only at higher pHs where the polymer is in its undoped form [11]. An alternative approach of the synthesis of water-soluble conducting polyaniline has been recently developed by introducing negatively charged polyelectrolyte into the reaction system [12–14]. In this case, the polyelectrolytes will emulsify the aniline monomer prior to the polymerization, and help to dope and dissolve the synthesized polymer. Most importantly, the polyelectrolyte molecules such as sulfonated polystyrene (SPS) will serve as template to promote a head-to-tail coupling during polymerization of aniline, which is necessary for the formation of conducting polyaniline [15]. With hazardous waste becoming increasingly expensive to treat, biochemical reactions are more attractive as alternative routes for synthesis of fine chemicals [16–18]. This way was also used in polymer chemistry [19]. Because horseradish peroxidase oxidizes aniline [15,20], this enzyme has been used in the synthesis of water-soluble polyaniline. The reaction was carried out in the presence of hydrogen peroxide as a reducing substrate and some polymeric templates having sulfonic and phosphoric groups. The advantage of the enzymatic approach compared to chemical one is that the synthesis has been carried out under mild conditions without generating toxic by-products. Unfortunately, horseradish peroxidase shows the low activity and stability at pH below 4.5 [15,21,22], i.e. in the pH interval where the polyelectrolyte complex between polyaniline (pKa of aniline is 4.63) and charged polymeric templates is formed. This results in a consumption of large amount of the peroxidase in the polymerization. For development of more technological processes alternative oxidoreductases capable effectively to polymerize aniline under acidic conditions should be used. To address this issue, a new biocatalyst, laccase, which can catalyze the oxidation of aniline in the presence of molecular dioxygen (O2 ), is introduced in synthesis of conducting polyaniline. Laccase (EC 1.10.3.2) is one of the oxygen oxidoreductases which is capable of oxidizing a lot of inorganic and organic substrates including aniline and its derivatives [23]. In our previous works, we have purified some laccases from Coriolus hirsutus, Coriolus zonatus and Cerena maxima [24–26]. Preliminary results have shown that the laccase C. hirsutus is active and stable under acidic conditions [25]. In this paper, we report a detailed investigation on the synthesis of water-soluble conducting SPS–polyaniline complex using laccase isolated from culture medium of C. hirsutus as a catalyst.

557

2. Experimental section 2.1. Materials Aniline, Na2 HPO4 and citric acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Aniline was purified by distillation before use. SPS (MW 70,000) was obtained from Aldrich (Milwaukee, WI) and used as available. Laccase was isolated from the culture fluid of C. hirsutus. The procedure for the purification was described elsewhere [25]. The specific activity of laccase measured toward catechol was not less than 350 units mg−1 of protein. 2.2. Laccase activity The activity of laccase C. hirsutus was usually determined spectrophotometrically in 100 mM acetate buffer (pH 4.2), containing 10 mM catechol as substrate, and the absorbance change at 410 nm was measured at 25 ◦ C for 60 s. The value of extinction coefficient for the oxidized products of catechol is 740 M−1 cm−1 . One unit of activity is defined as the amount of laccase oxidizing 1 ␮mol of substrate per min under standard conditions. Specific activity is expressed as units of activity per mg of protein. 2.3. Enzymatic polymerization of aniline The synthesis of SPS–polyaniline complexes was carried out usually in 0.1 M citrate–phosphate buffer, pH 3.5–4.4 with or without stirring at ambient temperature in humidity chamber. The concentration of monomer aniline and SPS in feed varied from 5 to 125 mM. The concentration of laccase in feed was usually 5.5 × 10−7 M. The reaction of laccase-catalyzed polymerization of aniline was evaluated using UV-Vis spectroscopy. 2.4. Spectroscopic methods The electronic spectra were recorded on a Shimadzu UV-2401 PC spectrophotometer. In each measurement distilled water was used as a control. The FTIR spectra were recorded on a spectrophotometer Nicolet Magna-750 using KBr pellets. 2.5. Cyclic voltammetry Electroanalytical experiments of polyaniline samples were performed using a BAS CV-50W Voltammetric Analyser (Bioanalytical System, USA). SPS–polyaniline was dissolved in a solution containing 0.01 M HCl and 0.1 M NaCl. The solution was loaded in a one-compartment electrochemical cell (volume 10 ml) consisted of an Ag/AgCl reference electrode, a platinum wire counter electrode and

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a 1 mm diameter platinum working electrode. The potential scan range was −100 to 1200 mV. Cyclic voltammograms were recorded with a scan rate of 100 mV s−1 . 2.6. Conductivity The electric conductivity of SPS–polyaniline was measured with a four-probe instrument. The sample was first dialyzed against 1 mM HCl and then freeze-dried. A round pellet was produced from the dried sample for the measurement of conductivity. 3. Results and discussion 3.1. Enzymatic polymerization Typically, peroxidase-catalyzed polymerization of conducting polyaniline is carried out at pH ∼ 4.0, which is not the optimal pH for HRP catalysis. Under these conditions HRP usually loses its catalytic activity a very short time period, which results in consumption of large amount of the enzyme during the polymerization reaction. Since laccase C. hirsutus is active and stable under acidic conditions [25], it will have a great advantage in comparison to HRP in the template-assisted polymerization of aniline. The laccase-catalyzed polymerization of aniline was studied in pH range from 3.5 to 4.4. The reaction was carried out at ambient temperature in the presence of SPS as mentioned in previous works [15]. The reactions were monitored with UV-Vis absorption spectroscopy. As shown in Fig. 1,

strong polaron absorption band at around 700–780 nm can be observed at pH 3.5, 3.7 and 3.9 indicating on the formation of conducting polyaniline. Therefore, we can conclude that laccase can catalyze the polymerization of aniline in the presence of SPS to form water-soluble conducting polyaniline under mild conditions compared to traditional chemical method [4,5]. Since the presence of O2 is necessary for laccase catalytic action, agitation should strongly affect the progress of reaction. This speculation was confirmed by comparative studying of the enzymatic polymerization with and without stirring (Fig. 2A). The reaction under constant stirring shows a higher reaction rate. The agitation appears to change the rate not only due to an increase of mass transfer of reacting compounds but maintenance of constant concentration of O2 consuming in the course of the polymerization. The stability of laccase was also investigated under the conditions of aniline polymerization. In this study, we also use the absorbance at 760 nm as an indicator of polyaniline production. Laccase was active in the solution for 4–5 days, demonstrating its high operational stability under the reaction conditions (Fig. 2B). 3.1.1. Effect of concentration of aniline and SPS on aniline polymerization The increase of aniline concentration up to 75 mM in feed results in the increase of the polymerization rate (Fig. 3). However, further increasing of aniline concentrations led to laccase inhibition by the substrate. Because of forming viscous final product solution in feed at 75 mM aniline concentration, preparative synthesis of polyelec-

1.5

absorbance

1.0

pH 3.7

pH 3.9

0.5

pH 3.5

pH 4.4

pH 4.2

0.0 300

400

500

600

700

800

900

wavelength, nm 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 M citrate–phosphate buffer; [aniline] = [SPS] = 6 mM; [laccase] = 5.5 × 10−7 M; the spectra were recorded 15 min after the polymerization initiation.

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Fig. 2. Kinetics of the polymerization reaction of aniline in the presence of SPS and laccase with (circles) and without (triangles) stirring. The experimental conditions: 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 out at 50 mM aniline. The effect of SPS concentration on the polymerization was also evaluated. It was shown that the increase of SPS concentration in feed results in the decrease of the rate of the aniline polymerization (Fig. 4). We have speculated that it was due to a change of oxygen concentration in the reaction medium in the presence of the template polymer. However, the measurement of oxygen concentration using a Clark’s electrode showed no difference in solutions with different

SPS concentration. Thus, the presence of SPS does not affect the oxygen concentration in the solution. The decrease of laccase activity with increasing of SPS concentration can also be considered as another possible reason for the change of reaction rate. To evaluate it, the effect of SPS concentration on the laccase activity measured against catechol, a specific substrate for laccase, was determined. However, the rate of catechol oxidation did not depend on the SPS concentration under experimental conditions used and, therefore, SPS does not affect the laccase activity.

absorbance at 760 nm

100

75

50

25

0

25

50

75

100

125

150

[aniline], mM Fig. 3. Effect of concentrations of aniline on rate of the template production of water-soluble SPS–polyaniline complexes by laccase. The experimental conditions: 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 polymerization initiation.

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Fig. 4. Effect of concentrations of SPS on rate of the template production of water-soluble SPS–polyaniline complexes by laccase. The experimental conditions: 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 polymerization initiation.

Comparison of chemical structures of aniline and catechol showed that in contrary of the latter compound aniline carries a positive charged group (–NH3 + ) and, therefore, may form ionic complexes with SPS under experimental conditions. At increasing SPS concentration an equilibrium between aniline, SPS and aniline–SPS complex is shifted toward the formation of the complex leading to removal of aniline from reaction mixture. Thus, we suggest that the decrease of aniline polymerization rate is connected with decrease of free aniline concentration in solution due to formation of aniline–SPS complexes. This conclusion allows us to suppose a mechanism of the template synthesis of polyaniline in the presence of laccase. At mixing of the reacting compounds a part of aniline molecules reacts with SPS forming their complexes, whereas the other part stays in a free state. After addition of the enzyme free aniline molecules are oxidized by the laccase and their radical products produced during the enzymatic step migrate to SPS–aniline complexes. There these radicals initiate template polymerization of aniline with formation of SPS–polyaniline complex. We suggest that some radicals 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 be significantly higher than that in volume. Even if some part of aniline molecules reacts in bulk, the oligomeric products formed produce immediately polyelectrolyte complexes with SPS. The proposed mechanism has been confirmed by UV-Vis spectroscopy detecting only existence of polyaniline bound with SPS. Further alignment of polyaniline chain appears to be carried out due to a reaction of aniline oligomers

stated in the complex with radical products formed by the enzyme. The data obtained permitted to compare the laccase from C. hirsutus and horseradish peroxidase as catalysts of aniline polymerization. Although both enzymes catalyze effectively the oxidative polymerization of aniline, the laccase shows higher operational stability under the reaction conditions. It should be emphasized also that in the contrary to the peroxidase which loses its activity in the presence of its oxidizing substrate (hydrogen peroxide) [27,28], the laccase is not inactivated by oxygen (the oxidizing agent of laccase). These features make the laccase more attractive for production of conducing polyaniline than horseradish peroxidase. 3.1.2. Characterization of water-soluble SPS–polyaniline complexes The solubility of the synthesized complex of SPS and polyaniline is strongly depended on the feed ratio of SPS to aniline. One day after initiation of the aniline polymerization all polyelectrolyte complexes obtained at different ratios of molar concentrations of repeating unit of SPS and aniline in feed (1:3, 3:5 and 1:1) were soluble in water. However, later for the two former complexes we detected the formation of precipitates. Only the polyaniline sample obtained at a ratio 1:1 of molar concentrations of repeating unit of SPS and aniline in feed was soluble completely. Such polyelectrolyte complex was stable at 4 and 25 ◦ C for 6 months as a minimum. This complex dialysed against 1 mM HCl and freeze-dried may be re-dissolved in aqueous solutions or N-methylpyrrolidone.

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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 with NaOH and HCl solutions. 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).

3.2. Doping and dedoping The SPS–polyaniline complex produced at the optimal conditions (pH 3.7 and a molar ratio of SPS and aniline of 1:1) has been characterized in detail. As seen in Fig. 5, electronic spectrum of the SPS–polyaniline complex under acidic conditions exhibits three characteristic absorption bands. The first absorption band at 320–360 nm arises from ␲–␲∗ electron transition within benzenoid segments. The second (400–420 nm) and third (760 nm) absorption bands are related to doping level and formation of polaron of the conducting form, respectively [29,30]. The former two peaks are combined into a single flat peak as described previously [31]. The shift of the position of the third peak as a function of pH of the reaction medium was observed at titration of the SPS–polyaniline complex by NaOH and HCl (Fig. 5). At decreasing pH from 3.7 to 2.9 the electronic spectrum did not change, whereas the titration of the polyaniline complex using sodium alkali changed completely the UV-Vis spectrum. The increase of the pH value in the polyaniline complex solution clove ionic bounds formed between the polymers, and the polyaniline become undoped that is reflected in change of UV-Vis spectra (Fig. 5). The transition point from doped to undoped form lays between pH 6.2 and 7.5. As pH values above the transition point (under alkaline conditions), the bands at 420 and 760 nm disappear, and a strong absorption at 556 nm begins to emerge. Previously the appearance of band at 550–580 nm was observed also for aqueous solutions of polyaniline-co-2-acrylamido-2-methyl-1-propanesulfonic

acid, polyelectrolyte complex of PANI and SPS and sulfonic acid ring-substituted PANI under strongly alkaline conditions [15,32,33]. Here it should be noted that for PANI dissolved in organic solvents the same band is observed at around 640 nm [1]. 3.3. FTIR The FTIR spectra of SPS and SPS–polyaniline complex are presented in Fig. 6. Comparison of these spectra showed the existence of some bands in the spectrum of the complex characteristic for polyaniline. The peaks at 1560 and 1488 cm−1 are assigned to a C=C ring stretching in quinoid and benzenoid units of polyaniline in doped form [13,34,35]. The sulfonic groups of SPS show characteristic bands at 1180 and 1042 cm−1 [32]. However, the formation of some ionic bonds between sulfonic groups of SPS and imine groups of polyaniline in the complex shifts this value to 1215 cm−1 . The presence of the peak at 1299 cm−1 characteristic for Caromatic –N stretching vibration confirms that polyaniline in the complex is in doped form, because for undoped polyaniline the same peak is revealed at 1310–1313 cm−1 [32,36]. The peaks at 799 and 880 cm−1 indicate head-to-tail coupling of the monomer with the formation of linear polymeric chains [35]. 3.4. Electroactivity Aqueous solution of the SPS–polyaniline complex in 0.01 M HCl, pH 2.0 was electrochemically active, as can be seen from its cyclic voltammogram in Fig. 7. For this

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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 of concentration of repeating unit of SPS and aniline of 1:1 ([aniline] = [SPS] = 50 mM).

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Fig. 7. Cyclic voltammetry of the SPS–polyaniline complex solution in 0.01N HCl, pH 2.0. Voltammogram was recorded with a scan rate of 100 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 cathodic sweep. The first cathodic peak is broad, not well resolved and pseudoreversible asymmetric with anodic peak observed in the potential range at 350–550 mV versus Ag/AgCl. The second cathodic irreversible peak has strong current maximum at potential about 50 mV. To compare the results obtained with those from the literature it should be noted that cyclic voltammograms for polyaniline produced enzymatically by laccase and horseradish peroxidase [15] or electrochemically [37] were not identical that gives to researchers broad possibilities for production of polyaniline with different electrochemical characteristics. The dc conductivity of the SPS–polyaniline complex was measured with a four-probe method. The conductivity value of the complex was 2 × 10−4 S cm−1 . Although this magnitude is not high in comparison with best samples of doped polyaniline (approximately several hundreds S cm−1 [1]), it is similar to those measured previously for water-soluble polyanilines produced by other methods [15,35]. It should be noted that in many cases, for polyanilines, being soluble and, hence, processable, is sometimes more important

than being highly conductive [10]. Thus, high solubility in aqueous solutions of the enzymatically obtained polyaniline complexes compensates their drawback connecting with its not enough high conductivity. Acknowledgments The authors thank Prof. Boris V. Lakshin for FTIR analysis, Dr. Elena V. Stepanova for the preparation of the enzyme preparation and Dr. Lynne Samuelson and Dr. Wei Lui for valuable discussions and conductivity measurements. This work was supported by the Russian Foundation of Basic Researches (Grant 02-04-48885). References [1] Pron A, Rannou P. Processable conjugated polymers: from organic semiconductors to organic metals and superconductors. Prog Polym Sci 2002;27:135–90. [2] MacDiamid AG. Synthetic metals: a novel role for organic polymers. Synth Met 2002;125:11–22.

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