European Polymer Journal 39 (2003) 23752381 www.elsevier.

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Immobilization of invertase and glucose oxidase in poly 2-methylbutyl-2-(3-thienyl) acetate/ polypyrrole matrices
Sonnur Is k a, Selmiye Alkan a, Levent Toppare Yusuf Yac b g
a

a,*

, Ioan Cianga

b,1

Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey b Department of Chemistry, Istanbul Technical University, 80626 Istanbul, Turkey

Received 29 October 2002; received in revised form 21 July 2003; accepted 24 July 2003

Abstract Immobilization of invertase and glucose oxidase in conducting polypyrrole and copolymers of poly 2-methylbutyl-2(3-thienyl) acetate with pyrrole were achieved via electrochemical method. Sodium dodecyl sulphate was found to be the most suitable supporting electrolyte. Maximum reaction rate, MichaelisMenten constant and optimum temperatures were determined for native and immobilized enzymes. Storage and operational stabilities of enzyme electrodes were also investigated. 2003 Elsevier Ltd. All rights reserved.
Keywords: Electropolymerization; Immobilization; Glucose oxidase; Invertase; Polypyrrole

1. Introduction Enzymes are protein molecules which serve to accelerate the chemical reactions of living cells. They speed up (bio)chemical reactions by lowering the energy of activation. Immobilized enzymes are preferred over the native ones owing to their multiple and repetitive use. In addition, the reaction product is not contaminated with the enzyme (especially useful in the food and pharmaceutical industries). Furthermore, the immobilized enzyme has a longer half-life and predictable decay rate [1]. Enzymes are immobilized by carrier binding (physical adsorption, ionic binding, covalent bonding), by crosslinking, adsorption or entrapment methods [2]. The entrapment of enzymes in conducting polymer matrices during electrochemical polymerization is attracting great
Corresponding author. Tel.: +90-312-210-3251; fax: +90312-210-1280. E-mail address: toppare@metu.edu.tr (L. Toppare). 1 On leave from Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania.
*

interest, because the method is simple, speedy, reliable and inexpensive. Moreover, enzyme molecules can be entrapped during electropolymerization in one step and polymer lm uniformly covers the surface of the substrate electrode of any shape or size. Furthermore, the lm thickness can be easily controlled by regulating the amount of charge passed. Immobilization procedure involves only the application of suitable potential on an electrode in appropriate aqueous solutions of monomers and enzymes [37]. In literature, several works were reported on the immobilization of enzymes in polypyrrole and its derivatives [811]. Invertase (EC 3.2.1.26) is produced from suitable commercial yeast strains grown on molasses, and unlike bacterial and fungal exoenzymes, it must be released by distribution of the cell wall. It is mainly used to hydrolyze sucrose (to glucose and fructose) in the preparation of invert sugar which has a lower crystallinity than sucrose at the high concentrations employed. Its use in confectionery thus ensures that the products remain fresh and soft even when kept for longer periods of time. Soluble invertase is used in the sweet industry in the

0014-3057/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0014-3057(03)00184-8

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production of articial honey, and a small extent in the industrial production of liquid sugar. Immobilization of invertase on corn grits [12], gelation [13], carbohydrate moieties [14] and polyelectrolytes [15] has been reported. Glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. In the foodstu industry glucose oxidase is employed for removing oxygen from soft drinks and canned foods, as well as glucose from egg products. A major clinical laboratory use of glucose oxidase is in glucose detection or assay in urine and blood. Therefore, construction of glucose electrode oers the possibility of continuously selfmonitoring of blood glucose levels by diabetics [16]. In this study, immobilizations of invertase and glucose oxidase by entrapment within polypyrrole (PPy) and poly 2-methylbutyl-2-(3-thienyl) acetate)/polypyrrole, PMBTA/PPy, copolymer matrices via electrochemical method were investigated. The synthesis and characterization of both PMBTA and PMBTA/PPy copolymer was described in detail previously [17]. Optimum temperature and kinetic parameters (Vmax , the maximum reaction rate and Km the anity of enzyme towards substrate) for the immobilized enzymes were examined. Furthermore, surface morphologies of enzyme entrapped lms were investigated. Operational and storage stabilities of the enzyme electrodes were determined.

O O

Galvanostatic Polymerization

O O

S MBTA

PMBTA

Scheme 2. Electrochemical polymerization of MBTA.

was carried out by constant current of 30 mA for 5 min. The solution in the cell purged with nitrogen for 3 min prior to polymerization (Scheme 2). 2.3. Enzyme immobilization Immobilization of invertase was achieved by electrochemical polymerization of pyrrole either on bare or PMBTA coated platinum electrodes. The electrolysis solution consisted of invertase (1.0 mg ml1 ), SDS (0.6 mg ml1 ) as the supporting electrolyte and acetate buer (pH 5.1). Electrochemical polymerization was carried out under constant potential of +1.0 V for 30 min at room temperature (a potentioscan Wenking POS-73 model potentiostat was used). The solution was purged with nitrogen during electrolysis. Afterwards, electrodes were washed with distilled water and stored in acetate buer at 4 C. Immobilization of glucose oxidase was performed with the same procedure as in the case of invertase except the electrolysis solution consisted of glucose oxidase (2 mg ml1 ) (Scheme 3). 2.4. Activity measurements of enzyme electrodes The activities of free and immobilized invertase were determined according to Nelson method [18]. Dierent concentrations of sucrose solution were preincubated for 10 min at 25 C. Then, enzyme electrode was placed into sucrose solutions for specic reaction times (2, 4, 6 min). After removing the electrode, 1 ml of aliquots were drawn and 1 ml of Nelson reagent was added to terminate the reaction. Then, tubes were placed in boiling water for 20 min. After cooling, 1 ml arsenomolybdate was added as the coloring agent. Next, the solutions were diluted to 7 ml with distilled water. Absorbances

2. Experimental 2.1. Materials Tetrabutylammonium tetrauoroborate, [CH3 (CH2 )3 ]4 NBF4 was obtained from Aldrich and dichloromethane from Merck. Invertase (E.C. 3.2.1.26) was purchased from Sigma. Pyrrole (Aldrich) was distilled before use and sodium dodecyl sulphate (SDS) (Aldrich) were used as received. Glucose oxidase (E.C. 1.13.4), peroxidase (E.C. 1.11.1.7), o-dianasidine, sulphuric acid and hydrogen peroxide were obtained from Merck. MBTA was synthesized by esterication of thiophene-3-acetic acid (Fluka) and (S)-(-)-2 methylbutanol (Aldrich) in the presence of p-toluene sulfonic acid, (PTSA) (Scheme 1). 2.2. Electrochemical polymerization of MBTA [CH3 (CH2 )3 ]4 NBF4 (0.65 g) and MBTA (50 mg) were dissolved in dichloromethane (10 ml). Polymerization

COOH S +

OH

PTSA benzene S MBTA

CO-O

Scheme 1. Synthesis of MBTA.

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H N O O Potentiostatic Polymerization S n H N x S n O O H N y

PMBTA

PMBTA/PPy

Scheme 3. Electrochemical polymerization of PMBTA in the presence of Py.

for the blank and the solutions were measured at 540 nm (a Shimadzu UV-1601 model spectrophotometer was used). For the free invertase activity determination, varying concentrations of sucrose solutions were preincubated for 10 min at 25 C and 0.1 ml of enzyme solution (0.01 mg/ml) was added. After the reaction for specic times, 1 ml Nelson reagent was added. Rest of the procedure was the same as in the case of invertase activity determination. The activity assay for free and immobilized glucose oxidase was carried out according to a modied version of Sigma Bulletin [19]. For that purpose, dierent concentrations of glucose solutions (2.550 mM) were prepared (in buer). Then, they were placed in water bath, shaken for 10 min at 25 C for preincubation. Enzyme electrode was then placed in glucose solution and the reaction was carried out for specic times. After removing the electrode, 0.5 ml of aliquots were drawn and 0.1 ml POD (60 U/ml), 2.4 ml o-dianisidine (21 mM) as the reducing agent were added. The reaction was stopped with the addition of 0.5 ml sulfuric acid (2.5 M). After mixing, absorbances for blank and the solution were measured at 530 nm. For the free enzyme, dierent concentrations of glucose solutions were prepared and preincubated in water bath for 10 min at 25 C. Then 0.1 ml glucose oxidase added and reacted for specic times. The rest of the procedure was the same as the enzyme electrode case. One unit of invertase activity was dened as amount of enzyme required to release 1 lmol glucose equivalent per minute at pH 5.1, 25 C. One unit of glucose oxidase activity was dened as the oxidation of 1 lmol of bD -glucose to D -gluconic acid and H2 O2 per minute at pH 5.1, 25 C.

bilization of invertase and glucose oxidase [57,20]. The activities of the invertase immobilized in SDS-system was found to be the best, however, for glucose oxidase all the electrolytes showed almost the same activity in the presence of dierent supporting electrolytes. The electrolysis times for immobilizations of invertase and glucose oxidase were dierent for dierent electrolytes; 30 min electrolysis with SDS, 120 min or more with both PTSA and NaPTS. Therefore, for the rest of the work, SDS was used as the supporting electrolyte for both enzymes due to the minimum time requirement for the immobilization. Because of using low concentration of supporting electrolytes for the enzyme entrapment during the immobilization, conductivities of enzyme immobilized lms were found to be in the order of 103 S/cm. 3.2. Morphology of immobilized enzymes The morphologies of enzyme entrapped lms were investigated by scanning electron microscopy (SEM) by JEOL JSM-6400. For the solution sides of the SDS doped PMBTA/PPy lm without enzyme (Fig. 1a), the cauliower-like structure which was usually observed for copolymers of pyrrole was dominant. For the invertase (Fig. 1b) and glucose oxidase (Fig. 1c) entrapped lms, the cauliower-like structure was signicantly damaged. Moreover, enzyme clusters were observed in the structure. The type of supporting electrolyte is most crucial according to our previous studies [21,22]. Since the anion of the electrolyte acts as the dopant ion for the conducting polymers/copolymers where the surface microstucture of the lm is aected. This eect in return plays another role in the eciency of the immobilization. This is due to the lack of cauliower morphology on the lm surface. The enzyme is not only embedded in the polymer but also trapped on the surface which makes them visible under SEM. 3.3. Eect of temperature on activity of enzymes

3. Results and discussion 3.1. Eect of supporting electrolytes on immobilization Dierent supporting electrolytes, p-toluene sulfonic acid (PTSA), sodium p-toluene sulfonate (NaPTS) and sodium dodecyl sulfate (SDS) were used for the immo-

The optimum temperatures for PPy/invertase (Fig. 2a), PPy/PMBTA/invertase (Fig. 2b) matrices prepared

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Fig. 1. SEM micrographs (10 l/cm) of (a) solution side of PPy/PMBTA matrix, (b) solution side of PPy/PMBTA/invertase matrix and (c) solution side of PPy/PMBTA/glucose oxidase matrix.

(a)

(b)

(c)

(d)

Fig. 2. Eect of temperature on enzyme electrodes. (a) PPy/invertase matrix, (b) PPy/PMBTA/invertase matrix, (c) PPy/glucose oxidase matrix and (d) PPy/PMBTA/glucose oxidase matrix.

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in the presence of SDS were found as 50 C. Invertase entrapped PMBTA/PPy electrode was more stable than

PPy electrode, because after reaching maximum activity at 50 C, PMBTA/PPy matrices have still high activity, however, PPy matrices lost almost all of its activity. The

Table 1 Kinetic constants for sucrose hydrolysis by free and immobilized invertase at 25 C and at optimum pH Km (mM) Free invertase PPy/invertase PPy/PMBTA/invertase
a b

Vmax (lmol/min) 82.3a 2.6b 6.1b

Table 2 Kinetic constant for glucose oxidation by free and immobilized glucose oxidase at 25 C and at optimum pH Km (mM) Free glucose oxidase PPy/glucose oxidase PMBTA/PPy/glucose oxidase
a b

59 53 87

Vmax (lmol/min) 16.8a 1.0b 0.8b

18.5 21.7 22

Per ml solution. Per electrode.

Per ml solution. Per electrode.

(a)

(b)

(c)

(d)
Fig. 3. Operational stability of (a) PPy/PMBTA/invertase matrix, (b) PPy/invertase matrix, (c) PPy/PMBTA/glucose oxidase matrix and (d) PPy/glucose oxidase matrix.

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(a)

(b)
Fig. 4. Storage stability of (a) PPy/PMBTA/invertase and (b) PPy/PMBTA/GOD.

optimum temperature for PPy/glucose oxidase (Fig. 2c), PPy/PMBTA/glucose oxidase electrodes prepared in SDS was also found to be 30 C (Fig. 2d). For free glucose oxidase, optimum temperature of about 30 C was reported [20]. This shows the similarity of microenvironments of the immobilized enzymes and the free enzymes.

3.5. Operational stability and shelf life of the enzyme electrodes To estimate the stability of electrodes in terms of repetitive uses, 20 successive measurements were performed at 25 C during one day for both enzymes (Fig. 3). For the immobilized invertase in PPy/PMBTA matrices (Fig. 3a) almost no change in their activity was observed. For the immobilized glucose oxidase with the same matrix (Fig. 3c), a small change in the activity was obtained. For the immobilized invertase in PPy matrices (Fig. 3b) small uctuations were observed compared to PMBTA matrices. However, the immobilized glucose oxidase in PPy matrices (Fig. 3d), revealed same behavior compared to the PPy/PMBTA matrices, in terms of activity and Km . Invertase entrapped in PPy/PMBTA matrices have storage stability (Fig. 4a) i.e., in 60 days only 7% of invertase lost its activity whereas in PPy matrices between 1 and 15 days it loses its activity to 40% then between the 15 and 30 days its activity remains constant and thereafter it further loses activity (Fig. 4b). Immobilization of glucose oxidase in these matrices has no storage stability, almost after one day glucose oxidase loses its activity.

3.4. Kinetic parameters of immobilized enzyme The kinetic studies were performed for free and immobilized enzymes at dierent concentrations of substrate. The apparent Michaelis constant (Km ) and maximum velocity Vmax were determined from LineweaverBurk plots [23]. For the invertase entrapped in PPy and PMBTA/PPy matrices, there is an increase in Km values compared to free invertase (Table 1). The increase in Km values (decreased anity) is due to electrostatic interactions between the substrate and matrix and also diusion eects. Enzymesubstrate complex formation becomes more dicult due to the structure of the conducting polymers (low porosity, rigid structure, etc.) for immobilized enzymes. However, Km values for immobilized glucose oxidase were comparable that of free enzyme (Table 2). It was concluded that immobilization did not cause any hindrance of enzyme towards its substrate. The decrease in the reaction rate Vmax of both immobilized invertase and glucose oxidase may be attributed to the restricted diusion of substrate to the enzyme. For the immobilized invertase, PMBTA/PPy matrices exhibited higher reaction rates than PPy matrices. However, for the immobilized glucose oxidase, reaction rate is almost the same for both matrices.

4. Conclusions This work shows that PMBTA/PPy copolymer can be used as enzyme immobilization matrix for invertase and glucose oxidase. SDS was found to be the best supporting electrolyte for the entrapments. Both enzymes were dispersed homogeneously in the electrode since they show appreciable activity with respect to free enzyme activity. Immobilization of invertase in PMBTA/

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PPy matrices is more feasible than in PPy matrices as regards to their optimum temperature and storage stability. Immobilization of glucose oxidase for both PMBTA/PPy and PPy matrices reveal almost the same stability. As to the stability, in PMBTA/PPy matrices, invertase immobilization is more feasible than that of glucose oxidase.

Acknowledgements This work is partially supported by TUBITAK research funds TBAG-2221 (102T116) and METU BAP2002-01-03-01.

References
[1] Mesing R. Immobilized enzymes for industrial reactors. Academic Press; 1995. [2] Chibata I. Immobilized enzymes. New York: Kodansha Ltd., Halsted Press; 1978. [3] Foulds NC, Lowe CR. J Chem Soc Faraday Trans 1986;82:1259. [4] Bartlett PN, Whitaker RG. J Electroanal Chem 1987; 224:27. [5] Selampnar F, Akbulut U, Ozden MY, Toppare L. Biomaterials 1997;18:1163. [6] Kzlyar N, Ozden MY, Toppare L, Yaci Y. Synth Met g 1999;104:45.

[7] Alkan S, Toppare L, Yac Y, Hepuzer Y. J Biomater Sci g Polym Edn 1999;10:1233. [8] Yasuzawa M, Niede T, Hirana T, Kunugi A. Sensors Actuat B: Chem 2000;66:77. [9] Almeida NF, Beckman EJ, Ataai MM. Biotechnol Bioeng 1993;42:1037. [10] Cosnier S, Lepellec A, Guidetti B, Rico-Lattes I. J Electroanal Chem 1998;449:165. [11] Diaz AF, Kanazawa KK, Gardini GP. J Chem Soc Chem Commun 1979:635. [12] Monsan P, Combes O. Biotechnol Bioeng 1984;26:347. [13] Akbulut U, Sungur S, Pekyardimci S. Macromol Rep 1991;28:239. [14] Marek M, Valentova O, Kas J. Biotechnol Bioeng 1984;26:1223. [15] Mansfeld J, Frster M, Schellenberger A, Dautzenberg H. o Enzyme Microb Technol 1991;13:240. [16] Home PD, Alberti KGM. Biosensors: fundamentals and applications. Oxford: Oxford Press; 1987. p. 723. [17] Levent A, Toppare L, Cianga I, Yagc Y. Macromol Chem Phys 2003;204:1118. [18] Nelson N. J Biol Chem 1994;153:375. [19] The enzymatic colorimetric determination of glucose, Sigma Technical Bulletin No 510, St Louis USA: Sigma Chemical Co.; 1983. [20] Tirkes S, Toppare L, Alkan S, Bakr U, Onen A, Yac Y. g Int J Biol Macromol 2002;30:81. [21] Alkan S, Toppare L, Yaci Y, Hepuzer Y. J Biomater Sci g Polym Ed 1999;10:1223. [22] Enginer R, Toppare L, Alkan S, Bakr U. React Funct Polym 2000;45:227. [23] Lineweawer H, Burk D. J Am Chem Soc 1934;56:658.