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polypyrrole block copolymers
REACTIVE & FUNCTIONAL POLYMERS
Reactive & Functional Polymers 57 (2003) 57–65
www.elsevier.com/locate/react
Immobilization of invertase and glucose oxidase in conducting H-type polysiloxane/polypyrrole block copolymers Altan G€ ursel a, Selmiye Alkan b, Levent Toppare a
b,*
, Yusuf Ya gcı
c
Department of Polymer Science and Technology, Middle East Technical University, 06531 Ankara, Turkey b Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey c Department of Chemistry, Istanbul Technical University, 80626 Istanbul, Turkey Received 24 October 2002; received in revised form 3 June 2003; accepted 25 July 2003
Abstract In this study, immobilizations of enzymes, invertase and glucose oxidase, were achieved in conducting copolymers of N-pyrrolyl terminated polydimethylsiloxane/polypyrrole (PDMS/PPy) matrices via electrochemical polymerization. The kinetic parameters, vmax (maximum reaction rate) and Km (substrate affinity), of both free and immobilized enzymes were determined. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate, on enzyme activity and film morphologies was examined. The optimum temperatures and operational stabilities of immobilized enzymes were determined. PDMS/PPy copolymer matrix was found to exhibit significantly enhanced properties compare to pristine polypyrrole in terms of relative enzyme activities, kinetic parameters and operational stabilities. Ó 2003 Published by Elsevier B.V. Keywords: Immobilization; Invertase; Glucose oxidase; Polysiloxane; Electropolymerization
1. Introduction Enzymes are the biocatalytically active entities upon which the metabolism of all living organism is based. They facilitate biochemical reactions by lowering the energy of activation [1]. An immobilized enzyme is one which has been attached to or enclosed by an insoluble support medium or one where the enzyme molecules have been cross-
*
Corresponding author. Tel.: +90-312-210-32-51; fax: +90312-210-12-80. E-mail address: [email protected] (L. Toppare). 1381-5148/$ - see front matter Ó 2003 Published by Elsevier B.V. doi:10.1016/j.reactfunctpolym.2003.07.004
linked to each other, without loss of catalytic activity [2]. Immobilized enzymes provide several operational advantages over free enzyme. The reusability without significant loss of activity, enhanced stability, controlled product formation, ease of separation of enzyme from reaction products and high enzymatic activity in a small volume are the attractive features of immobilized systems. Invertase (EC 3.2.1.26) catalyses the conversion of sucrose to glucose and fructose. This hydrolysis reaction, yielding sugar mixtures, is widely used in the production of noncrystallizing creams, in making jam and artificial honey. Invertase has
58
A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65 H OH N CH2 CH CH2 N CH2
3
CH3 Si O CH3
n
CH3 Si CH2 CH3
3
H OH N CH2 CH CH2 N
Scheme 1. N-pyrrolyl terminated polydimethylsiloxane (Mn ¼ 1500).
rather lower probability of achieving commercial use in immobilized form; however, it is one of the most studied of all enzymes because of being a model enzyme for immobilization of more expensive and more applicable enzymes. Immobilization of invertase on gelatin [3], carbohydrate moieties [4], polyelectrolytes [5], covalent attachment onto the surface modified polyaniline [6], entrapment in polyethylene vinyl alcohol [7], organo-functionalized polysiloxane copolymers [8] were performed. Entrapment of invertase by electrochemical method in several conducting copolymer matrices were described in detail [9–12]. Glucose oxidase (EC 1.1.3.4) catalyses the oxidation of b-D -glucose to D -glucono-1,5-lactone and hydrogen peroxide using molecular oxygen as the electron acceptor. Glucose oxidase is widely used for the determination of glucose in body fluids and in removing residual glucose and oxygen from beverages and foodstuffs. The immobilization of glucose oxidase in various matrices was reported in literature previously. Several biosensors detecting glucose concentration were constructed. Covalent attachment of glucose oxidase to copolymers of methyl and glycidyl methacrylate [13], immobilization of glucose oxidase at platinum electrode by electrochemical polymerization of phenol and its derivatives [14], entrapment in 2-hydroxyethyl methacrylate [15], encapsulation in SiO2 gels [16], entrapment of glucose oxidase in polypyrrole [17–20] were described in detail. The synthesis and characterization of both pristine PDMS and copolymers of PDMS with pyrrole have been described previously [21]. Compared to pristine polypyrrole, the copolymer exhibited better conductivity, thermal and mechanical properties. Therefore, in this work, PDMS/PPy copolymer was further tested as an enzyme immobilization matrix. In this study, immobilizations of invertase and glucose oxidase were achieved in copolymers of Npyrrolyl terminated polydimethylsiloxane/polypyr-
role (PDMS/PPy). The kinetic parameters of both free and immobilized enzymes were determined. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate, on enzyme activity were examined. The optimum temperatures, operational stabilities and film morphologies of immobilized enzymes were determined Scheme 1.
2. Experimental 2.1. Material and technique Invertase (EC 3.2.1.26) Type V, glucose oxidase (EC 1.1.3.4) Type II-S, peroxidase (EC 1.11.1.7) Type II, o-dianisidine were purchased from Sigma and used as received without further purification. Pyrrole (Merck) was distilled before use and stored at 4 °C. Sodium dodecyl sulfate and p-toluene sulfonic acid were obtained from Sigma and used as received. Sulfuric acid, hydrogen peroxide and sodium hydroxide were supplied from Merck. Potentioscan Wenking POS-73 and ST-88 potentiostats, Shimadzu UV-1601 model spectrophotometer and JEOL JSM-6400 model scanning electron microscope (SEM) were used. 2.2. Immobilizations of invertase and glucose oxidase in PDMS/PPy matrices Immobilization of invertase or glucose oxidase was performed in a typical three electrode cell containing platinum (Pt) working and counter electrodes and a silver/silver ion reference electrode by constant potential electrolysis at room temperature. Electrolysis solution consists of invertase (1 mg/ml) or glucose oxidase (2 mg/ml); SDS (0.6 mg/ml) or PTSA (21 mg/ml) as the supporting electrolyte, pyrrole (0.02 M) and acetate buffer (pH 5). Immobilization of enzymes were carried out on either bare or PDMS coated electrodes. After electrolysis, enzyme electrodes were
A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65
washed with distilled water in order to remove both the supporting electrolyte and unbound enzyme and kept in acetate buffer at 4 °C. 2.3. Determination of invertase activity Determination of immobilized and free invertase activities was performed by using Nelson method [22]. For free invertase activity determination, sucrose solutions were placed in test tubes, incubated at 25 °C. After addition of 0.1 ml enzyme solution (0.01 mg/ml), enzyme and substrate were allowed to react for specific times and at the end of each period, 1 ml Nelson reagent was added to terminate the reaction. After 20 min in hot water bath, the solutions were kept in ice bath for 10 min. Next, 1 ml arsenomolibdate reagent and 7 ml distilled water were added to the solution for color formation. The absorbances of solutions were measured at 540 nm. For immobilized invertase activity determination, enzyme electrode was placed in sucrose solution and after specific reaction times, the electrode was removed and 1 ml aliquot was drawn and 1 ml Nelson reagent was added. The rest of the procedure was the same as the free invertase activity determination which was described above. One unit invertase activity (EU) was defined as the amount of enzyme required to produce 1 lmol glucose from sucrose per minute at pH 5 and 25 °C. 2.4. Determination of glucose oxidase activity The activity determination was performed by using a modified version of Sigma Bulletin [23]. For free glucose oxidase activity determination, glucose solutions were placed in test tubes and incubated at 25 °C. After addition of 0.1 ml enzyme solution, enzyme and substrate were allowed to react for specific times. Then, 0.1 ml POD (60 U/ml)––to catalyze the reaction of hydrogen peroxide––and 2.4 ml o-dianisidine (0.21 mM)––as the coloring agent––were added. The reaction was terminated with the addition of 0.5 ml 2.5 M sulfuric acid then spectrophotometric measurements were performed at 530 nm. For the determination of the activity of immobilized enzyme, the reaction was started by placing enzyme electrode in glucose solution and after specific reaction times, 0.5 ml of
59
aliquots were drawn, the rest of the procedure was applied mentioned for free enzyme activity. H2 O2 standard calibration curve was used in order to define enzyme activity. One unit of glucose oxidase activity (EU) was defined as the amount of enzyme required to produce 1 lmol of D -gluconic acid and H2 O2 per minute at pH 5 and 25 °C. 2.5. Determination of kinetic parameters In order to determine maximum velocity of the reaction (vmax ) and the Michaelis–Menten constant (Km ) for each electrode, activity assay was applied for different concentrations of sucrose (invertase assay) and glucose (glucose oxidase assay). 2.6. Determination of optimum temperature Optimum temperatures for immobilized invertase and glucose oxidase were determined by changing incubation temperature between 10 to 70 °C and 15 to 45 °C, respectively, while keeping substrate concentration constant. 2.7. Morphologies of the films The morphologies of polymer films both with and without enzyme were investigated. After peeling off the films from electrode and washing with buffer solution for several times to remove unbound enzyme and supporting electrolyte from the surface of film, SEM analyses were performed. 2.8. Operational stabilities Operational stabilities of immobilized enzymes in polymer films were tested (at optimum activity assay conditions) by performing 30 activity assays during one day.
3. Results and discussion 3.1. Optimization of electrolysis conditions Immobilization of invertase in both PPy and PDMS/PPy matrices was carried out in acetate
60
A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65
buffer containing enzyme, pyrrole and supporting electrolyte, namely, SDS or PTSA by constant potential electrolysis. Entrapment of invertase was achieved in about 60 min in the presence of SDS (0.6 mg/ml) in acetate buffer (pH 5). A homogeneous black film with ca. 20 lm thickness was obtained at the end of electrolysis period for both matrices. In the presence of PTSA as the supporting electrolyte, immobilization of invertase was performed after optimization of both PTSA concentration and the pH of the electrolysis medium. The activities of immobilized invertase at varying PTSA concentration were presented in Table 1. At low PTSA concentrations, no film formation was observed, although immobilization was allowed to proceed for longer electrolysis times. The use of high concentration of PTSA yielded high quality polymer film; however, no detectable enzyme activity was obtained in this case. This can be attributed to the fact that performing electrolysis in such a high PTSA concentration causes the denaturation of the enzyme due to acidic character of the supporting electrolyte. Therefore, for the immobilization of invertase, the pH of the medium was adjusted to 5 by the addition of NaOH (0.1 M) to the acetate buffer containing high concentration of PTSA (21 mg/ml). As far as the activities of invertase immobilized in the presence of SDS and PTSA were compared, it was found that the activity in SDS solution (1.98 EU/electrode) was about seven times as high as the activity in PTSA solution (0.28 EU/electrode). High activity of invertase in the presence of SDS was due to its surfactant property. This provides the ease of diffusion of enzyme to the working electrode [9–11]. Table 1 Relative activities of the immobilized invertase at varying PTSA concentrations PTSA (mg/ml)
pH
Activity (EU/electrode)
0.6 1 3 6 12 30
4.8 4.7 4.3 3.6 1.7 1.2
No coating No coating 0.11 0.12 No activity No activity
In glucose oxidase immobilization, almost similar activities were obtained in the presence of different supporting electrolytes. In terms of electrolysis times required for the formation of enzyme entrapped films with good quality and sufficient thickness, SDS was found to be better compared to PTSA [24]. Therefore, in this study, glucose oxidase immobilization was carried out in the presence of SDS (0.6 mg/ml) in acetate buffer (pH 5). 3.2. Morphologies of the films The morphologies of PDMS/PPy copolymers with and without invertase were investigated. The free standing films were obtained in the following media; SDS/acetate buffer, PTSA/acetate buffer and PTSA/deionized water systems. The solution sides of both SDS (Fig. 1a) and PTSA (Fig. 1b) doped film in acetate buffer exhibited cauliflower like structure which is usually observed for the copolymers produced with pyrrole. However, the morphology of the copolymers prepared in deionized water was quite different (Fig. 1c). The incorporation of polysiloxane units was observable and leading to the destruction of common polypyrrole morphology. This behavior was also obtained in the previous study [25]. The morphology of enzyme entrapped copolymers was significantly changed especially for the solution sides of the films. In the SDS doped copolymers, the destruction in cauliflower-like structure and the formation of enzyme clusters were observed for both invertase (Fig. 2a) and glucose oxidase (Fig. 2c) entrapped films. PTSA doped invertase entrapped films exhibited cauliflower-like structures without significant damage when together with enzyme clusters (Fig. 2b). These results revealed that SDS provides the best electrolytic immobilization conditions for the invertase which are compatible with the kinetic studies and relative activity measurements. It was also concluded that for the enzyme entrapped film, the type of supporting electrolyte had strong influence on the film morphology as well as the type of enzyme. At the solution sides of PPy and PDMS/PPy films with both invertase and glucose oxidase
A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65
Fig. 1. Morphology of solution sides of PDMS/PPy film (a) SDS doped (in acetate buffer), (b) PTSA doped (in acetate buffer) and (c) PTSA doped (in deionized water).
immobilized, the destruction of cauliflower-like structure was observed, being more drastic in PDMS/PPy matrices compared to pristine PPy [9,11] which may provide suitable medium for the substrate diffusion. Electrode side of copolymer films had almost smooth surfaces with some remaining traces originating from mainly supporting electrolyte (Fig. 3a). Although significantly altered film morphology was observed for the electrode side of the invertase entrapped films with damaged structure and dominant enzyme clusters (Fig. 3b), glucose
61
Fig. 2. Morphology of solution sides of PDMS/PPy film in acetate buffer (a) invertase entrapped (SDS doped), (b) invertase entrapped (PTSA doped) and (c) glucose oxidase entrapped (SDS doped).
oxidase entrapped films exhibited rather smooth surfaces with little hollows without large defects or irregularities and without enzyme clusters in the structure (Fig. 3c). 3.3. Kinetic parameters Kinetic parameters, Km and vmax , of the free and immobilized enzymes were determined at constant temperature, pH while varying substrate concentrations. For both invertase and glucose oxidase,
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A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65 Table 2 Kinetic parameters of free and immobilized invertase
a
Free invertase PPy/invertase PDMS/PPy/SDS PDMS/PPy/PTSA a
Fig. 3. Morphology of electrode sides of PDMS/PPy film in acetate buffer (SDS doped) (a) without enzyme, (b) invertase entrapped and (c) glucose oxidase entrapped.
enzyme activity profile was found to follow the Michaelis–Menten kinetics [26]. Therefore, Km and vmax were obtained from the Lineweaver–Burk plots [27]. The Km and vmax values for free and immobilized invertase were tabulated in Table 2. Km value for PDMS/PPy copolymer (SDS doped) matrix was comparable with that of free invertase and it showed better affinity than both pristine PPy and PDMS/PPy (PTSA doped) matrices. Although in previous invertase immobilization studies [9,11], the affinity of immobilized enzyme was found to be
Km (mM)
vmax (EU/electrode)
24.3 52.9 26.0 46.6
82.3 2.6 4.2 0.4
EU/ml.
significantly lower (higher Km ), in this study, similar affinity of immobilized (SDS doped) and free enzyme was further proved the suitability of PDMS/PPy copolymer as the enzyme immobilization matrix indicating that being entrapped in the polymer matrices did not cause any hindrance of the enzyme towards its substrate. Furthermore, immobilization of enzyme in PDMS/PPy (SDS doped) copolymer matrix further resulted in the enhancement of the affinity compared to pristine PPy matrix which may be attributed to the fact that this matrix provided convenient immobilization medium for this enzyme. PDMS/PPy (PTSA doped) copolymer matrix exhibited higher Km value which revealed the decreased affinity of enzyme to its substrate in the presence of PTSA. The acidic property of PTSA may cause denaturation of enzyme during immobilization procedure. However, vmax values were significantly altered by means of immobilization which is in agreement with the previous studies [9–11]. The dramatic decrease in vmax of the enzyme substrate reaction for the immobilized systems may be caused by the difficulty of substrate diffusion owing to the polymer structure (insufficient porosity, rigidity due to the long polypyrrole chains) of the enzyme entrapped matrices. Glucose oxidase entrapped in PDMS/PPy and PPy matrices exhibited similar Km values which were also comparable with that of free enzyme (Table 3). However, the maximum reaction rate Table 3 Kinetic parameters of free and immobilized glucose oxidase
Free glucose oxidasea PPy/GOD PDMS/PPy/GOD a
EU/ml.
Km (mM)
vmax (EU/electrode)
18.5 21.7 20.4
16.8 1.0 1.1
A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65
was lowered by means of immobilization due to the reasons explained for the case of the immobilized invertase described above. 3.4. Effect of incubation temperature on enzyme activity The variation of invertase activity with temperature was investigated for entrapped invertase in both SDS and PTSA doped PDMS/PPy copolymer matrices. The results for a temperature range from 10 to 70 °C were presented in Fig. 4a and b. For SDS doped matrix, immobilized invertase exhibited high activity for a temperature range between 40 and 60 °C and for PTSA doped matrix between 40 and 50 °C, instead of a specific temperature. The optimum temperatures for free invertase and pristine PPy were determined previously as 50 and 60 °C, respectively [9,11]. High enzyme activity over a temperature range rather than a definite temperature revealed the stability against temperature in PDMS/PPy matrix.
The maximum activity was observed at 30 °C for both glucose oxidase entrapped PPy and PDMS/PPy copolymer electrodes (Fig. 5) as well as for the free enzyme [24]. This result may be due to the similar microenvironments of the immobilized and free glucose oxidase. 3.5. Operational stability In order to determine the operational stabilities of immobilized enzymes, activity assays were performed during the same day. For both invertase entrapped in PDMS/PPy and pristine PPy matrices (Fig. 6), activities of enzyme electrode remained constant with small fluctuations for 30 successive measurements. Furthermore, PDMS/PPy copolymer matrix exhibited higher stability compared to PPy matrix. For glucose oxidase entrapped matrices (Fig. 7), after initial loss in activity in first five assays, it remained constant. This may occurred due to the leaching of the loosely bound enzyme from the matrix during the early stage of the activity assay.
relative activity
relative activity
100 80 60 40 20 0 0
20
40
60
(a)
100 80 60 40 20 0
80
0
0
20
40
60
80
0
(b)
temperature C
63
temperature C
relative activity
relative activity
Fig. 4. Effect of the incubation temperature on the invertase activity of (a) PDMS/PPy electrode (SDS doped) (b) PDMS/PPy electrode (PTSA doped).
100 80 60 40 20
100
60 40 20 0
0 0
(a)
80
10
20
30
40 0
temperature C
50
0
(b)
10
20
30
40
50
0
temperature C
Fig. 5. Effect of the incubation temperature on the glucose oxidase activity of SDS doped (a) PPy electrode (b) PDMS/PPy electrode
A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65
relative activity
64
Acknowledgements
100 90 80 70 60 50 40 30 20 10 0
pdms/ppy/sds/inv
This work is partially supported by grants TBAG-2221 (102T116) and BAP-2002-01-03-01.
ppy/sds/inv 0
5
10
15
20
25
30
assay number
relative activity
Fig. 6. Effect of repeated use on the activity of immobilized invertase.
100 90 80 70 60 50 40 30 20 10 0
pdms/ppy/sds/god ppy/sds/god 0
5
10
15
20
25
30
assay number
Fig. 7. Effect of repeated use on the activity of immobilized glucose oxidase.
4. Conclusions Immobilizations of invertase and glucose oxidase were successfully achieved for PDMS/PPy copolymer and PPy matrices. Invertase entrapped copolymer matrices exhibited comparable Km values with free enzyme. Moreover, enhanced stability against temperature and better operational stability were observed for invertase entrapped in PDMS/PPy matrix compared to pristine PPy matrix. Furthermore, significantly changed morphologies in the presence of invertase were observed for both solution and electrode side of the films. As the supporting electrolyte, SDS found to provide better immobilization medium for invertase than PTSA. For the immobilized glucose oxidase, although PDMS/PPy matrix showed similar characteristics in terms of temperature stability and substrate affinity, it showed better relative activity than PPy matrices. It was concluded that PDMS/PPy copolymer is a suitable immobilization matrix for both enzymes since it was porous enough to permit diffusion of substrate into enzyme entrapped film as well as sufficiently strong to prevent to leakage of enzyme from the matrix.
References [1] W. Hartmeier, Immobilized Biocatalysts, Springer-Verlag, Berlin, 1986. [2] T. Palmer, Understanding Enzymes, Prentice-Hall & Ellis Horwood, London, 1991. [3] U. Akbulut, S. Sungur, S. Pekyardimci, Macromol. Rep. 28 (1991) 239. [4] M. Marek, O. Valentova, J. Kas, Biotechnol. Bioeng. 26 (1984) 1223. [5] J. Mansfeld, M. F€ orster, A. Schellenberger, H. Dautzenberg, Enzyme Microb. Technol. 13 (1991) 240. [6] Y. Chen, E.T. Kang, K.G. Neoh, K.L. Tan, Eur. Polym. J. 36 (2000) 2095. [7] K. Imai, T. Shiami, K. Uchida, M. Miya, Biotechnol. Bioeng. 28 (1986) 198. [8] D.L. Venton, E. Gudipati, Biochim. Biophys. Acta 1250 (1995) 117. € zden, L. Toppare, [9] F. Selampinar, U. Akbulut, M.Y. O Biomaterials 18 (1997) 1163. € zden, Y. [10] N. Kizilyar, U. Akbulut, L. Toppare, M.Y. O Yagci, Synth. Met. 104 (1999) 45. [11] S. Alkan, L. Toppare, Y. Yagci, Y. Hepuzer, J. Biomater. Sci. Polym. Ed. 10 (1999) 1223. [12] R. Erginer, L. Toppare, S. Alkan, U. Bakir, React. Funct. Polym. 45 (2000) 227. [13] C.E. Hall, D. Datta, E.A.H. Hall, Anal. Chim. Acta 323 (1996) 87. [14] P.N. Bartlett, P. Tebbutt, C.H. Tyrrell, Anal. Chem. 64 (1992) 138. [15] I. G€ ursel, V.N. Hasirci, Biomaterials 13 (1992) 150. [16] U. K€ unzelmann, H. B€ ottcher, Sens. Actuators B 38–39 (1997) 222. [17] N.C. Foulds, C.R. Lowe, Anal. Chem. 60 (1988) 2473. [18] N.F. Almeida, E.J. Beckman, M.M. Ataai, Biotechnol. Bioeng. 42 (1993) 1037. [19] M. Yasuzawa, T. Nieda, T. Hirano, A. Kunugi, Sens. Actuators B 66 (2000) 77. [20] K. Ramanathan, S. Annapoorni, A. Kumar, B.D. Malhotra, J. Mater. Sci. Lett. 15 (1996) 124. [21] E. Kalaycıoglu, L. Toppare, Y. Yagcı, V. Harabagiu, M. Pintela, R. Ardelean, B.C. Simionescu, Synth. Met. 97 (1998) 7. [22] N. Nelson, J. Biol. Chem. 153 (1944) 375. [23] Sigma Technical Bulletin No. 510. The Enzymatic Colorimetric Determination of Glucose. Sigma Chemical Co., St Louis, MO, USA, 1983. € nen, Y. [24] S. Tirkesz, L. Toppare, S. Alkan, U. Bakır, A. O Yagcı, Int. J. Biol. Macromol. 30 (2002) 81.
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[26] L. Michaelis, M.L. Menten, Biochem. Z. 49 (1913) 333. [27] H. Lineweaver, D. Burk, J. Am. Chem. Soc. 56 (1934) 658.
Reactive & Functional Polymers 57 (2003) 57–65
www.elsevier.com/locate/react
Immobilization of invertase and glucose oxidase in conducting H-type polysiloxane/polypyrrole block copolymers Altan G€ ursel a, Selmiye Alkan b, Levent Toppare a
b,*
, Yusuf Ya gcı
c
Department of Polymer Science and Technology, Middle East Technical University, 06531 Ankara, Turkey b Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey c Department of Chemistry, Istanbul Technical University, 80626 Istanbul, Turkey Received 24 October 2002; received in revised form 3 June 2003; accepted 25 July 2003
Abstract In this study, immobilizations of enzymes, invertase and glucose oxidase, were achieved in conducting copolymers of N-pyrrolyl terminated polydimethylsiloxane/polypyrrole (PDMS/PPy) matrices via electrochemical polymerization. The kinetic parameters, vmax (maximum reaction rate) and Km (substrate affinity), of both free and immobilized enzymes were determined. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate, on enzyme activity and film morphologies was examined. The optimum temperatures and operational stabilities of immobilized enzymes were determined. PDMS/PPy copolymer matrix was found to exhibit significantly enhanced properties compare to pristine polypyrrole in terms of relative enzyme activities, kinetic parameters and operational stabilities. Ó 2003 Published by Elsevier B.V. Keywords: Immobilization; Invertase; Glucose oxidase; Polysiloxane; Electropolymerization
1. Introduction Enzymes are the biocatalytically active entities upon which the metabolism of all living organism is based. They facilitate biochemical reactions by lowering the energy of activation [1]. An immobilized enzyme is one which has been attached to or enclosed by an insoluble support medium or one where the enzyme molecules have been cross-
*
Corresponding author. Tel.: +90-312-210-32-51; fax: +90312-210-12-80. E-mail address: [email protected] (L. Toppare). 1381-5148/$ - see front matter Ó 2003 Published by Elsevier B.V. doi:10.1016/j.reactfunctpolym.2003.07.004
linked to each other, without loss of catalytic activity [2]. Immobilized enzymes provide several operational advantages over free enzyme. The reusability without significant loss of activity, enhanced stability, controlled product formation, ease of separation of enzyme from reaction products and high enzymatic activity in a small volume are the attractive features of immobilized systems. Invertase (EC 3.2.1.26) catalyses the conversion of sucrose to glucose and fructose. This hydrolysis reaction, yielding sugar mixtures, is widely used in the production of noncrystallizing creams, in making jam and artificial honey. Invertase has
58
A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65 H OH N CH2 CH CH2 N CH2
3
CH3 Si O CH3
n
CH3 Si CH2 CH3
3
H OH N CH2 CH CH2 N
Scheme 1. N-pyrrolyl terminated polydimethylsiloxane (Mn ¼ 1500).
rather lower probability of achieving commercial use in immobilized form; however, it is one of the most studied of all enzymes because of being a model enzyme for immobilization of more expensive and more applicable enzymes. Immobilization of invertase on gelatin [3], carbohydrate moieties [4], polyelectrolytes [5], covalent attachment onto the surface modified polyaniline [6], entrapment in polyethylene vinyl alcohol [7], organo-functionalized polysiloxane copolymers [8] were performed. Entrapment of invertase by electrochemical method in several conducting copolymer matrices were described in detail [9–12]. Glucose oxidase (EC 1.1.3.4) catalyses the oxidation of b-D -glucose to D -glucono-1,5-lactone and hydrogen peroxide using molecular oxygen as the electron acceptor. Glucose oxidase is widely used for the determination of glucose in body fluids and in removing residual glucose and oxygen from beverages and foodstuffs. The immobilization of glucose oxidase in various matrices was reported in literature previously. Several biosensors detecting glucose concentration were constructed. Covalent attachment of glucose oxidase to copolymers of methyl and glycidyl methacrylate [13], immobilization of glucose oxidase at platinum electrode by electrochemical polymerization of phenol and its derivatives [14], entrapment in 2-hydroxyethyl methacrylate [15], encapsulation in SiO2 gels [16], entrapment of glucose oxidase in polypyrrole [17–20] were described in detail. The synthesis and characterization of both pristine PDMS and copolymers of PDMS with pyrrole have been described previously [21]. Compared to pristine polypyrrole, the copolymer exhibited better conductivity, thermal and mechanical properties. Therefore, in this work, PDMS/PPy copolymer was further tested as an enzyme immobilization matrix. In this study, immobilizations of invertase and glucose oxidase were achieved in copolymers of Npyrrolyl terminated polydimethylsiloxane/polypyr-
role (PDMS/PPy). The kinetic parameters of both free and immobilized enzymes were determined. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate, on enzyme activity were examined. The optimum temperatures, operational stabilities and film morphologies of immobilized enzymes were determined Scheme 1.
2. Experimental 2.1. Material and technique Invertase (EC 3.2.1.26) Type V, glucose oxidase (EC 1.1.3.4) Type II-S, peroxidase (EC 1.11.1.7) Type II, o-dianisidine were purchased from Sigma and used as received without further purification. Pyrrole (Merck) was distilled before use and stored at 4 °C. Sodium dodecyl sulfate and p-toluene sulfonic acid were obtained from Sigma and used as received. Sulfuric acid, hydrogen peroxide and sodium hydroxide were supplied from Merck. Potentioscan Wenking POS-73 and ST-88 potentiostats, Shimadzu UV-1601 model spectrophotometer and JEOL JSM-6400 model scanning electron microscope (SEM) were used. 2.2. Immobilizations of invertase and glucose oxidase in PDMS/PPy matrices Immobilization of invertase or glucose oxidase was performed in a typical three electrode cell containing platinum (Pt) working and counter electrodes and a silver/silver ion reference electrode by constant potential electrolysis at room temperature. Electrolysis solution consists of invertase (1 mg/ml) or glucose oxidase (2 mg/ml); SDS (0.6 mg/ml) or PTSA (21 mg/ml) as the supporting electrolyte, pyrrole (0.02 M) and acetate buffer (pH 5). Immobilization of enzymes were carried out on either bare or PDMS coated electrodes. After electrolysis, enzyme electrodes were
A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65
washed with distilled water in order to remove both the supporting electrolyte and unbound enzyme and kept in acetate buffer at 4 °C. 2.3. Determination of invertase activity Determination of immobilized and free invertase activities was performed by using Nelson method [22]. For free invertase activity determination, sucrose solutions were placed in test tubes, incubated at 25 °C. After addition of 0.1 ml enzyme solution (0.01 mg/ml), enzyme and substrate were allowed to react for specific times and at the end of each period, 1 ml Nelson reagent was added to terminate the reaction. After 20 min in hot water bath, the solutions were kept in ice bath for 10 min. Next, 1 ml arsenomolibdate reagent and 7 ml distilled water were added to the solution for color formation. The absorbances of solutions were measured at 540 nm. For immobilized invertase activity determination, enzyme electrode was placed in sucrose solution and after specific reaction times, the electrode was removed and 1 ml aliquot was drawn and 1 ml Nelson reagent was added. The rest of the procedure was the same as the free invertase activity determination which was described above. One unit invertase activity (EU) was defined as the amount of enzyme required to produce 1 lmol glucose from sucrose per minute at pH 5 and 25 °C. 2.4. Determination of glucose oxidase activity The activity determination was performed by using a modified version of Sigma Bulletin [23]. For free glucose oxidase activity determination, glucose solutions were placed in test tubes and incubated at 25 °C. After addition of 0.1 ml enzyme solution, enzyme and substrate were allowed to react for specific times. Then, 0.1 ml POD (60 U/ml)––to catalyze the reaction of hydrogen peroxide––and 2.4 ml o-dianisidine (0.21 mM)––as the coloring agent––were added. The reaction was terminated with the addition of 0.5 ml 2.5 M sulfuric acid then spectrophotometric measurements were performed at 530 nm. For the determination of the activity of immobilized enzyme, the reaction was started by placing enzyme electrode in glucose solution and after specific reaction times, 0.5 ml of
59
aliquots were drawn, the rest of the procedure was applied mentioned for free enzyme activity. H2 O2 standard calibration curve was used in order to define enzyme activity. One unit of glucose oxidase activity (EU) was defined as the amount of enzyme required to produce 1 lmol of D -gluconic acid and H2 O2 per minute at pH 5 and 25 °C. 2.5. Determination of kinetic parameters In order to determine maximum velocity of the reaction (vmax ) and the Michaelis–Menten constant (Km ) for each electrode, activity assay was applied for different concentrations of sucrose (invertase assay) and glucose (glucose oxidase assay). 2.6. Determination of optimum temperature Optimum temperatures for immobilized invertase and glucose oxidase were determined by changing incubation temperature between 10 to 70 °C and 15 to 45 °C, respectively, while keeping substrate concentration constant. 2.7. Morphologies of the films The morphologies of polymer films both with and without enzyme were investigated. After peeling off the films from electrode and washing with buffer solution for several times to remove unbound enzyme and supporting electrolyte from the surface of film, SEM analyses were performed. 2.8. Operational stabilities Operational stabilities of immobilized enzymes in polymer films were tested (at optimum activity assay conditions) by performing 30 activity assays during one day.
3. Results and discussion 3.1. Optimization of electrolysis conditions Immobilization of invertase in both PPy and PDMS/PPy matrices was carried out in acetate
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buffer containing enzyme, pyrrole and supporting electrolyte, namely, SDS or PTSA by constant potential electrolysis. Entrapment of invertase was achieved in about 60 min in the presence of SDS (0.6 mg/ml) in acetate buffer (pH 5). A homogeneous black film with ca. 20 lm thickness was obtained at the end of electrolysis period for both matrices. In the presence of PTSA as the supporting electrolyte, immobilization of invertase was performed after optimization of both PTSA concentration and the pH of the electrolysis medium. The activities of immobilized invertase at varying PTSA concentration were presented in Table 1. At low PTSA concentrations, no film formation was observed, although immobilization was allowed to proceed for longer electrolysis times. The use of high concentration of PTSA yielded high quality polymer film; however, no detectable enzyme activity was obtained in this case. This can be attributed to the fact that performing electrolysis in such a high PTSA concentration causes the denaturation of the enzyme due to acidic character of the supporting electrolyte. Therefore, for the immobilization of invertase, the pH of the medium was adjusted to 5 by the addition of NaOH (0.1 M) to the acetate buffer containing high concentration of PTSA (21 mg/ml). As far as the activities of invertase immobilized in the presence of SDS and PTSA were compared, it was found that the activity in SDS solution (1.98 EU/electrode) was about seven times as high as the activity in PTSA solution (0.28 EU/electrode). High activity of invertase in the presence of SDS was due to its surfactant property. This provides the ease of diffusion of enzyme to the working electrode [9–11]. Table 1 Relative activities of the immobilized invertase at varying PTSA concentrations PTSA (mg/ml)
pH
Activity (EU/electrode)
0.6 1 3 6 12 30
4.8 4.7 4.3 3.6 1.7 1.2
No coating No coating 0.11 0.12 No activity No activity
In glucose oxidase immobilization, almost similar activities were obtained in the presence of different supporting electrolytes. In terms of electrolysis times required for the formation of enzyme entrapped films with good quality and sufficient thickness, SDS was found to be better compared to PTSA [24]. Therefore, in this study, glucose oxidase immobilization was carried out in the presence of SDS (0.6 mg/ml) in acetate buffer (pH 5). 3.2. Morphologies of the films The morphologies of PDMS/PPy copolymers with and without invertase were investigated. The free standing films were obtained in the following media; SDS/acetate buffer, PTSA/acetate buffer and PTSA/deionized water systems. The solution sides of both SDS (Fig. 1a) and PTSA (Fig. 1b) doped film in acetate buffer exhibited cauliflower like structure which is usually observed for the copolymers produced with pyrrole. However, the morphology of the copolymers prepared in deionized water was quite different (Fig. 1c). The incorporation of polysiloxane units was observable and leading to the destruction of common polypyrrole morphology. This behavior was also obtained in the previous study [25]. The morphology of enzyme entrapped copolymers was significantly changed especially for the solution sides of the films. In the SDS doped copolymers, the destruction in cauliflower-like structure and the formation of enzyme clusters were observed for both invertase (Fig. 2a) and glucose oxidase (Fig. 2c) entrapped films. PTSA doped invertase entrapped films exhibited cauliflower-like structures without significant damage when together with enzyme clusters (Fig. 2b). These results revealed that SDS provides the best electrolytic immobilization conditions for the invertase which are compatible with the kinetic studies and relative activity measurements. It was also concluded that for the enzyme entrapped film, the type of supporting electrolyte had strong influence on the film morphology as well as the type of enzyme. At the solution sides of PPy and PDMS/PPy films with both invertase and glucose oxidase
A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65
Fig. 1. Morphology of solution sides of PDMS/PPy film (a) SDS doped (in acetate buffer), (b) PTSA doped (in acetate buffer) and (c) PTSA doped (in deionized water).
immobilized, the destruction of cauliflower-like structure was observed, being more drastic in PDMS/PPy matrices compared to pristine PPy [9,11] which may provide suitable medium for the substrate diffusion. Electrode side of copolymer films had almost smooth surfaces with some remaining traces originating from mainly supporting electrolyte (Fig. 3a). Although significantly altered film morphology was observed for the electrode side of the invertase entrapped films with damaged structure and dominant enzyme clusters (Fig. 3b), glucose
61
Fig. 2. Morphology of solution sides of PDMS/PPy film in acetate buffer (a) invertase entrapped (SDS doped), (b) invertase entrapped (PTSA doped) and (c) glucose oxidase entrapped (SDS doped).
oxidase entrapped films exhibited rather smooth surfaces with little hollows without large defects or irregularities and without enzyme clusters in the structure (Fig. 3c). 3.3. Kinetic parameters Kinetic parameters, Km and vmax , of the free and immobilized enzymes were determined at constant temperature, pH while varying substrate concentrations. For both invertase and glucose oxidase,
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A. G€ursel et al. / Reactive & Functional Polymers 57 (2003) 57–65 Table 2 Kinetic parameters of free and immobilized invertase
a
Free invertase PPy/invertase PDMS/PPy/SDS PDMS/PPy/PTSA a
Fig. 3. Morphology of electrode sides of PDMS/PPy film in acetate buffer (SDS doped) (a) without enzyme, (b) invertase entrapped and (c) glucose oxidase entrapped.
enzyme activity profile was found to follow the Michaelis–Menten kinetics [26]. Therefore, Km and vmax were obtained from the Lineweaver–Burk plots [27]. The Km and vmax values for free and immobilized invertase were tabulated in Table 2. Km value for PDMS/PPy copolymer (SDS doped) matrix was comparable with that of free invertase and it showed better affinity than both pristine PPy and PDMS/PPy (PTSA doped) matrices. Although in previous invertase immobilization studies [9,11], the affinity of immobilized enzyme was found to be
Km (mM)
vmax (EU/electrode)
24.3 52.9 26.0 46.6
82.3 2.6 4.2 0.4
EU/ml.
significantly lower (higher Km ), in this study, similar affinity of immobilized (SDS doped) and free enzyme was further proved the suitability of PDMS/PPy copolymer as the enzyme immobilization matrix indicating that being entrapped in the polymer matrices did not cause any hindrance of the enzyme towards its substrate. Furthermore, immobilization of enzyme in PDMS/PPy (SDS doped) copolymer matrix further resulted in the enhancement of the affinity compared to pristine PPy matrix which may be attributed to the fact that this matrix provided convenient immobilization medium for this enzyme. PDMS/PPy (PTSA doped) copolymer matrix exhibited higher Km value which revealed the decreased affinity of enzyme to its substrate in the presence of PTSA. The acidic property of PTSA may cause denaturation of enzyme during immobilization procedure. However, vmax values were significantly altered by means of immobilization which is in agreement with the previous studies [9–11]. The dramatic decrease in vmax of the enzyme substrate reaction for the immobilized systems may be caused by the difficulty of substrate diffusion owing to the polymer structure (insufficient porosity, rigidity due to the long polypyrrole chains) of the enzyme entrapped matrices. Glucose oxidase entrapped in PDMS/PPy and PPy matrices exhibited similar Km values which were also comparable with that of free enzyme (Table 3). However, the maximum reaction rate Table 3 Kinetic parameters of free and immobilized glucose oxidase
Free glucose oxidasea PPy/GOD PDMS/PPy/GOD a
EU/ml.
Km (mM)
vmax (EU/electrode)
18.5 21.7 20.4
16.8 1.0 1.1
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was lowered by means of immobilization due to the reasons explained for the case of the immobilized invertase described above. 3.4. Effect of incubation temperature on enzyme activity The variation of invertase activity with temperature was investigated for entrapped invertase in both SDS and PTSA doped PDMS/PPy copolymer matrices. The results for a temperature range from 10 to 70 °C were presented in Fig. 4a and b. For SDS doped matrix, immobilized invertase exhibited high activity for a temperature range between 40 and 60 °C and for PTSA doped matrix between 40 and 50 °C, instead of a specific temperature. The optimum temperatures for free invertase and pristine PPy were determined previously as 50 and 60 °C, respectively [9,11]. High enzyme activity over a temperature range rather than a definite temperature revealed the stability against temperature in PDMS/PPy matrix.
The maximum activity was observed at 30 °C for both glucose oxidase entrapped PPy and PDMS/PPy copolymer electrodes (Fig. 5) as well as for the free enzyme [24]. This result may be due to the similar microenvironments of the immobilized and free glucose oxidase. 3.5. Operational stability In order to determine the operational stabilities of immobilized enzymes, activity assays were performed during the same day. For both invertase entrapped in PDMS/PPy and pristine PPy matrices (Fig. 6), activities of enzyme electrode remained constant with small fluctuations for 30 successive measurements. Furthermore, PDMS/PPy copolymer matrix exhibited higher stability compared to PPy matrix. For glucose oxidase entrapped matrices (Fig. 7), after initial loss in activity in first five assays, it remained constant. This may occurred due to the leaching of the loosely bound enzyme from the matrix during the early stage of the activity assay.
relative activity
relative activity
100 80 60 40 20 0 0
20
40
60
(a)
100 80 60 40 20 0
80
0
0
20
40
60
80
0
(b)
temperature C
63
temperature C
relative activity
relative activity
Fig. 4. Effect of the incubation temperature on the invertase activity of (a) PDMS/PPy electrode (SDS doped) (b) PDMS/PPy electrode (PTSA doped).
100 80 60 40 20
100
60 40 20 0
0 0
(a)
80
10
20
30
40 0
temperature C
50
0
(b)
10
20
30
40
50
0
temperature C
Fig. 5. Effect of the incubation temperature on the glucose oxidase activity of SDS doped (a) PPy electrode (b) PDMS/PPy electrode
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relative activity
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Acknowledgements
100 90 80 70 60 50 40 30 20 10 0
pdms/ppy/sds/inv
This work is partially supported by grants TBAG-2221 (102T116) and BAP-2002-01-03-01.
ppy/sds/inv 0
5
10
15
20
25
30
assay number
relative activity
Fig. 6. Effect of repeated use on the activity of immobilized invertase.
100 90 80 70 60 50 40 30 20 10 0
pdms/ppy/sds/god ppy/sds/god 0
5
10
15
20
25
30
assay number
Fig. 7. Effect of repeated use on the activity of immobilized glucose oxidase.
4. Conclusions Immobilizations of invertase and glucose oxidase were successfully achieved for PDMS/PPy copolymer and PPy matrices. Invertase entrapped copolymer matrices exhibited comparable Km values with free enzyme. Moreover, enhanced stability against temperature and better operational stability were observed for invertase entrapped in PDMS/PPy matrix compared to pristine PPy matrix. Furthermore, significantly changed morphologies in the presence of invertase were observed for both solution and electrode side of the films. As the supporting electrolyte, SDS found to provide better immobilization medium for invertase than PTSA. For the immobilized glucose oxidase, although PDMS/PPy matrix showed similar characteristics in terms of temperature stability and substrate affinity, it showed better relative activity than PPy matrices. It was concluded that PDMS/PPy copolymer is a suitable immobilization matrix for both enzymes since it was porous enough to permit diffusion of substrate into enzyme entrapped film as well as sufficiently strong to prevent to leakage of enzyme from the matrix.
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