SYNTHESIS AND CHARACTERIZATION OF CONDUCTING
COPOLYMER OF BIS(4-(3-THIENYL
ETHYLENE)-OXYCARBONYL)DIAZOBENZENE WITH PYRROLE AND ITS
APPLICATION IN ENZYME IMMOBILIZATION.
M.Ak, H.B. Yildiz & L. Toppare
Middle East Technical University, Ankara, 06531, TURKEY
ABSTRACT:Bis(4-(3-thienyl ethylene)-oxycarbonyl)diazobenzene (TDAZO) was synthesized via the reaction between 4,4/-azodibenzoyl chloride and 3-thiophene ethanol. Conducting copolymer of TDAZO with pyrrole (Py), Poly[TDAZO-co-Py], was synthesized using sodium dodecyl sulfate as the supporting electrolyte via constant potential electrolyses. Invertase was immobi-lized in Poly[TDAZO-co-Py] copolymer. Immobilization of enzymes was carried out by the entrapment of the enzyme in conduct-ing polymer matrices durconduct-ing electrochemical polymerization of pyrrole through thiophene moieties of the TDAZO. Immobilization of the enzyme was achieved by application of 1.0 V constant potential on a platinum electrode for 30 min. Optimum conditions for this electrode, such as pH, temperature, and kinetic parameters (Km and Vmax), were investigated. The operational stability was also studied.
Keywords: conducting polymer, enzyme immobilization, invertase
1 INTRODUCTION
Thiophene is one of the most studied heterocycles (Barbarella te al 2005): it is easy to process, chemically stable and its synthetic applications have been a constant matter of investigation for the last six or seven decades. The interest in this het-erocycle has spread from early dye chemistry (King et al 1949) to modern drug design (Wu et al 2004), electronic and optoelectronic devices (Halik et al 2003 and Rost et al 2004), biodiagnostics (Dore et al 2004), block copolymer self assembled superstructures, and conductivity based sensory devices (Vrizerna et al 2003 and Yu et al 2004). Recent years, the generation of anisotropy induced by linearly polarized light in polymeric systems containing azobenzene moieties has attracted in-creasing attention because of the practical applica-tions such as waveguide and optical storage (Wu et al 1999 and Lanzi et al 2004). These polymeric systems can also be interesting for enzyme en-trapment applications.
Enzymes are biological catalysts that increase the rate of chemical reactions taking place within liv-ing cells by lowerliv-ing the energy of activation, without themselves appearing in the reaction prod-ucts. Unlike most inorganic catalysts, enzymes are generally soluble and unstable; thus, these organics can be used only once in solutions. Enzymes may be used in industry in free or immobilized forms (Palmer 1995).
Immobilization of enzymes makes heterogeneous catalysis possible, which has great advantages: it is possible to use a single batch of enzymes repeti-tively and to stop the reaction by physical removal
of the immobilized enzyme from the solution. Also, in many cases, the enzyme is stabilized by bonding. Additional advantages include easy ana-lyte determination in complex mixtures and use of small sample volumes. The enzyme will still be ac-tive and largely uncontaminated, so it can be used again. Also, due to the longer life, predictable de-cay rate and elimination of reagent preparation are further advantages of immobilization (Yildiz et al 2005a,b).
Invertase, known as -froctofuranosidase (E.C. 3.2.1.26), catalyzes the hydrolytic breakdown of sucrose to glucose and fructose. The mixture of these products has a lower crystallinity than su-crose at high concentrations. The use of invertase hence ensures that the products remain fresh and soft even when kept for a long time. Therefore, it is widely used in the production of artificial honey and to a small extent in the industrial production of liquid sugar (Yildiz et al 2005c). The immobilized form of invertase has so far only been employed experimentally, since the soluble enzyme is avail-able at little cost; however, the use of invertase in the entrapment process sheds light on the immobi-lization of expensive enzymes in conducting polymer matrices.
In this study bis(4-(3-thienyl ethylene)-oxycarbonyl)diazobenzene (TDAZO) was synthe-sized via the reaction between 4,4/-azodibenzoyl chloride and 3-thiophene ethanol. Conducting co-polymer of TDAZO with pyrrole,
Poly(TDAZO-co-Py), was synthesized using sodium dodecyl
sul-fate as the supporting electrolyte via constant po-tential electrolyses. The immobilization of inver-tase was done via entrapment within Poly(TDAZO-co-Py) characterized using IR spec-troscopy. Then, optimum conditions for these
elec-trodes, such as pH, temperature, and kinetic pa-rameters (Km and Vmax), were investigated. The operational stability was also studied.
2 EXPERIMENTAL 2.1 Materials
Sodium hydroxide, 4-nitrobenzoic acid, glucose, acetic acid, thionyl chloride, triethylamine, n-hexane, 3-thiophene ethanol were purchased from Aldrich and used without further purification. In-vertase, -froctofuranosidase (E.C. 3.2.1.26), and sodium dodecyl sulfate (SDS) were purchased from Sigma. Pyrrole (Merck) was distilled and stored at 4°C. For the preparation of the Nelson Reagent, sodium carbonate (Riedel de Haen), so-dium potassium tartarate (Riedel de Haen), soso-dium bicarbonate (Merck), and sodium sulfate (Merck), and for the preparation of the arsenomolybdate re-agent; ammonium heptamolybdate (Merck) and sodium arsenate (Merck) were used as received.
2.2 Equipment
For the electrochemical synthesis, a potentioscan Wenking POS-73 model potentiostat, a Shimadzu model FT-IR spectrophotometer for the characteri-zation of conducting copolymers, and a Shimadzu UV-1601 model spectrophometer for enzyme ac-tivity measurements were used.
2.3 Synthesis of bis(4-(3-thienyl
ethylene)-oxycarbonyl)diazobenzene (TDAZO)
A sodium hydroxide solution (25 g in 150 ml) was added to 4-nitrobenzoic acid (6 g, 37 mmol) and heated at 60 oC. A solution of glucose (50 g in 80 ml of water) was added slowly at this temperature with occasional shaking, the reaction mixture was then cooled to ambient temperature and stirred un-til orange crystals were formed. The mixture was acidified with dilute acetic acid and filtered. After washed with water, diacid was dissolved in hot po-tassium carbonate solution to get orange colored solution. This solution was concentrated to get or-ange crystals of potassium salt of diacid. On acidi-fying with dilute acetic acid, 9 g (86%) of orange colored 4,4/-azodibenzoic acid (Francisca and Kanna 1999) was obtained. FT-IR (KBr): 3600– 3000 cm-1 (m, br), 1691 cm-1 (s), 1600 cm-1 (s), 1579 cm-1 (s), 1426 cm-1 (s), 1300– 1200 cm-1 (s, br), 868 cm-1 (s), 773 (s) cm-1.
4,4/-azodibenzoic acid (2 g, 7.4 mmol) were placed into a 50 ml round-bottomed flask containing 8 ml of thionyl chloride and 0.1 ml triethylamine. The mixture was heated on an oil bath up to 60 oC, un-til the suspension mixture was converted to a clear
solution. Then, the solution was stirred over night at room temperature. Unreacted thionyl chloride was removed under reduced pressure and the resi-due was washed with dry n-hexane two times to leave 2.1 g (93%) of red crystals of 4,4/ -azodibenzoyl chloride (Faghihi and Hagibeygi 2003); mp 177–179 oC, FT-IR (KBr): 1776 (s), 1732 (m, br), 1595 (m), 1408 (m), 1195 (s, br),885 (s), 804 (m), 646 (m) cm-1
Mixture of 2.4 g (1.98 mL, 21 mmol) 3-thiophene ethanol and 2.02 g (2.77 mL, 20 mmol) dry triethyl amine (TEA) in a two necked round bottom flask equipped with a reflux condenser and a dropping funnel, 10 mmol (3.07 g) of 4,4/-azodibenzoic acid dissolved in 100 mL of dry chloroform were added dropwise with stirring. The reaction was then kept at a reflux for 3 days with continuous stirring. Upon the completion of the reaction, the reaction mixture was washed with distilled water and dried over CaCl2. After removal of solvent evaporation,
the residue was extracted with methanol to remove unreacted thiophene ethanol and 4,4/-azodibenzoic acid. Then residue was purified by reprecipitation three times from THF solution to hexane and solid product was obtained. Yield %75, mp: 157 oC FT-IR (KBr): 1786 (s), 1711 (s), 1600 (m), 1459 (m), 1408 (m), 1216 (s, br), 852 (s), 774 (m), 691 (m) cm-1
Scheme1. Synthesis of the bis(4-(3-thienyl ethylene)-oxycarbonyl)diazobenzene (TDAZO)
2.4 Synthesis of Poly(TDAZO-co-PPy)
1% solution of (w/v) of TDAZO was prepared in dichloromethane. Pyrrole was polymerized elec-trochemically on platinum (Pt) electrode that was previously coated with TDAZO. SDS was used as the supporting electrolyte in water, and electro-polymerization yielded a black film on the elec-trode after 30 min by applying 1.0 V against the Ag/Ag+ reference electrode.
O Cl N N O Cl 4,4/ - Azodibenzoyl chloride S CH2CH2OH + S CH2CH2O C O N N C O OCH2CH2 S Chloroform TEA TDAZO
2.5 Immobilization of invertase in
Poly(TDAZO-co-Py)
Electropolymerization was performed in a three compartment cell. This cell has Pt as the working and counter electrodes Ag/Ag+(0.001 M) reference electrode. Electrolysis solution consists of 10 mL acetate buffer (50 mM pH = 5.0) 0.6 mg/mL inver-tase, 0.6mg/mL SDS, and 40 L pyrrole. The working electrode was coated with TDAZO from its dichloromethane solution. Electrolyses were done at 1.0V constant potential on the potentiostat for 30 min.
2.6 Determination of enzyme activity
For immobilized invertase, different concentrations of sucrose in buffer solution (acetate buffer pH = 5.0) were placed in test tubes and put in a water bath at 25°C for 10 min. After preincubation, en-zyme electrodes were immersed in test tubes and shaken in the water bath for 2-6 min.
Aliquots (1.0 mL) of these solutions were with-drawn, and 1.0 mL Nelson's Reagent was added.The tubes were then placed in boiling water for 20 min. Samples were cooled in an ice-bath, and 1.0 mL of arsenomolybdate solution was added to the tubes and mixed by vortexing. Fi-nally, 7.0 mL of distilled water was added to each of the test tubes. After mixing, absorbance was measured at 540 nm using a Shimadzu UV-1601 model spectrophotometer.
2.7 Determination of optimum pH and
optimum temperature
The reaction temperature was changed between 10°C and 80°C while sucrose concentration was kept constant at about 10 Km for each system. For pH optimization at 25°C, the pH of the reaction medium was changed between pH 2 and pH 9 while sucrose concentration was kept constant at about 10 Km for each system. The activities were determined as previously described.
3 RESULTS AND DISCUSSION
3.1 FTIR characterization
Poly(TDAZO-co-Py)
The FTIR spectral characteristics of the copolymer were discussed together with that of the monomer. TDAZO showed a characteristic, intense peak at 1711 cm-1, which belongs to C=O stretching vibra-tions. Two peaks at 2948 cm-1 and 2867 cm-1 corre-spond to aliphatic methylene stretchings.The peak at 1251 cm-1 indicates C−O–C ester group
vibra-tions while the peak at 740 cm-1 is the result of aromatic C−Hα stretching of thiophene units. FTIR spectrum of P(TDAZO-co-Py) showed in-tense peaks at 1044, 1121 and 1127 cm-1 which be-longs to dopant ions. Two peaks at 2924 cm-1 and 2854 cm-1 correspond to aliphatic methylene stretching. A characteristic peak 1690 cm-1 belong-ing to carbonyl group of TDAZO was also ob-served. These results prove copolymerization.
3.2 Kinetic parameter of immobilized enzyme The maximum reaction rate, Vmax, and Michaelis-Menten constant, Km were obtained from Lineweaver-Burk plot.
Table 1. Kinetic parameters for free and immobilized inver-tase
Reaction rate decreased sharply since enzyme was entrapped in a matrice. Poly(TDAZO-co-Py) ma-trice shows a smaller Km value than free invertase. A smaller Km value than free enzyme indicates that this matrice provides a microenvironment that is more suitable than the one in the solution.
3.3 Effect of pH on enzyme activity
Due to the denaturation, effect of changes in pH on the enzyme structure becomes important. The maximum activity was obtained at pH 4.6 for the free enzyme (Kizilyar et al., 1999). The maximum pH was found to be 6.0 for the Poly(TDAZO-co-Py) (Figure 1). The optimum pH was shifted to-wards the alkaline side when compared with the free enzyme because of the presence of dopant ions in the matrice. The copolymer matrice gave the high stability at lower pHs, such as pH 3-5. There-fore, the copolymer matrice can be used reliably at low pH values for enzyme reactions.
0 20 40 60 80 100 0 2 4 6 8 10 pH R e la ti v e E n z y m e A c ti v ity
Figure 1. Effect of pH on invertase activity immobilized in TDAZO/PPy
3.4 Effect of temperature on enzyme activity The effect of temperature between 10 and 80°C on enzyme activity was investigated, and it is illus-trated in Figure 2. The maximum temperature for the free enzyme was found to be 50°C (Erginer et al.2000). Maximum enzyme activity for the matri-ces was at 40°C,. Although the copolymer matrice at low temperatures showed higher stability, at high temperatures the enzyme activity for this ma-trice reduces rapidly.
0 20 40 60 80 100 0 20 40 60 80 100 Tem perature (C) R e la ti v e E n z y m e A c ti v ity
Figure 2. Effect of incubation temperature on invertase activ-ity in Poly[TDAZO-co-Py]
3.5 Operational stability of the enzyme
electrode
It is tried to estimate the stability of the electrodes in terms of repetitive uses. In 40 successive meas-urements, we observed very small fluctuations for copolymer matrice. The results are shown in Fig-ure 4. The enzyme activities were almost stable during 40 experiments performed at 25°C in 1 day.
0 20 40 60 80 100 0 10 20 30 40 50 Assay Number Rel a ti ve E n z y m e Act ivi ty
Figure 3. Operational Stability of Poly[TDAZO-co-Py]
4 CONCLUSION
Bis(4-(3-thienylethylene)-oxycarbonyl) diazoben-zene (TDAZO) was synthesized by means of the reaction between 4,4/-azodibenzoyl chloride and 3-thiophene ethanol. Conducting copolymer of TDAZO with pyrrole, Poly(TDAZO-co-Py), was synthesized using sodium dodecyl sulfate as the supporting electrolyte via constant potential elec-trolyses and characterized via IR spectroscopy. The immobilization of invertase was done via en-trapment within Poly(TDAZO-co-Py). This study shows that Poly(TDAZO-co-Py) electrodes can be used for the immobilization of invertase, based on kinetic studies, temperature and pH optimization and stability studies.
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