Vous êtes sur la page 1sur 7

European Polymer Journal 39 (2003) 2375–2381


Immobilization of invertase and glucose oxidase

in poly 2-methylbutyl-2-(3-thienyl) acetate/
polypyrrole matrices
Sonnur Isßık a, Selmiye Alkan a, Levent Toppare a,*
, Ioan Cianga b,1
Yusuf Ya gcı b
Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey
Department of Chemistry, Istanbul Technical University, 80626 Istanbul, Turkey
Received 29 October 2002; received in revised form 21 July 2003; accepted 24 July 2003

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, Michaelis–Menten constant and optimum temperatures
were determined for native and immobilized enzymes. Storage and operational stabilities of enzyme electrodes were also
Ó 2003 Elsevier Ltd. All rights reserved.

Keywords: Electropolymerization; Immobilization; Glucose oxidase; Invertase; Polypyrrole

1. Introduction interest, because the method is simple, speedy, reliable

and inexpensive. Moreover, enzyme molecules can be
Enzymes are protein molecules which serve to accel- entrapped during electropolymerization in one step and
erate the chemical reactions of living cells. They speed polymer film uniformly covers the surface of the sub-
up (bio)chemical reactions by lowering the energy of strate electrode of any shape or size. Furthermore, the
activation. Immobilized enzymes are preferred over the film thickness can be easily controlled by regulating the
native ones owing to their multiple and repetitive use. In amount of charge passed. Immobilization procedure
addition, the reaction product is not contaminated with involves only the application of suitable potential on an
the enzyme (especially useful in the food and pharma- electrode in appropriate aqueous solutions of monomers
ceutical industries). Furthermore, the immobilized en- and enzymes [3–7]. In literature, several works were re-
zyme has a longer half-life and predictable decay rate [1]. ported on the immobilization of enzymes in polypyrrole
Enzymes are immobilized by carrier binding (physical and its derivatives [8–11].
adsorption, ionic binding, covalent bonding), by cross- Invertase (EC is produced from suitable
linking, adsorption or entrapment methods [2]. The en- commercial yeast strains grown on molasses, and unlike
trapment of enzymes in conducting polymer matrices bacterial and fungal exoenzymes, it must be released by
during electrochemical polymerization is attracting great distribution of the cell wall. It is mainly used to hydro-
lyze sucrose (to glucose and fructose) in the preparation
Corresponding author. Tel.: +90-312-210-3251; fax: +90- of invert sugar which has a lower crystallinity than su-
312-210-1280. crose at the high concentrations employed. Its use in
E-mail address: toppare@metu.edu.tr (L. Toppare). confectionery thus ensures that the products remain
On leave from ‘‘Petru Poni’’ Institute of Macromolecular fresh and soft even when kept for longer periods of time.
Chemistry, Iasi, Romania. Soluble invertase is used in the sweet industry in the
0014-3057/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
2376 S. Isßık et al. / European Polymer Journal 39 (2003) 2375–2381

production of artificial 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. O
O Polymerization
Glucose oxidase catalyzes the oxidation of glucose to O O
gluconic acid and hydrogen peroxide. In the foodstuff
industry glucose oxidase is employed for removing ox-
ygen from soft drinks and canned foods, as well as
S S n
glucose from egg products. A major clinical laboratory
use of glucose oxidase is in glucose detection or assay in MBTA PMBTA
urine and blood. Therefore, construction of glucose Scheme 2. Electrochemical polymerization of MBTA.
electrode offers the possibility of continuously self-
monitoring of blood glucose levels by diabetics [16].
In this study, immobilizations of invertase and glucose was carried out by constant current of 30 mA for 5 min.
oxidase by entrapment within polypyrrole (PPy) and The solution in the cell purged with nitrogen for 3 min
poly 2-methylbutyl-2-(3-thienyl) acetate)/polypyrrole, prior to polymerization (Scheme 2).
PMBTA/PPy, copolymer matrices via electrochemical
method were investigated. The synthesis and character- 2.3. Enzyme immobilization
ization of both PMBTA and PMBTA/PPy copolymer
was described in detail previously [17]. Optimum tem- Immobilization of invertase was achieved by elec-
perature and kinetic parameters (Vmax , the maximum trochemical polymerization of pyrrole either on bare or
reaction rate and Km the affinity of enzyme towards PMBTA coated platinum electrodes. The electrolysis
substrate) for the immobilized enzymes were examined. solution consisted of invertase (1.0 mg ml1 ), SDS (0.6
Furthermore, surface morphologies of enzyme en- mg ml1 ) as the supporting electrolyte and acetate buffer
trapped films were investigated. Operational and storage (pH 5.1). Electrochemical polymerization was carried
stabilities of the enzyme electrodes were determined. 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
2. Experimental
with nitrogen during electrolysis. Afterwards, electrodes
were washed with distilled water and stored in acetate
2.1. Materials
buffer at 4 °C. Immobilization of glucose oxidase was
performed with the same procedure as in the case of
Tetrabutylammonium tetrafluoroborate, [CH3 -
invertase except the electrolysis solution consisted of
(CH2 )3 ]4 NBF4 was obtained from Aldrich and dichlo-
glucose oxidase (2 mg ml1 ) (Scheme 3).
romethane from Merck. Invertase (E.C. was
purchased from Sigma. Pyrrole (Aldrich) was distilled
before use and sodium dodecyl sulphate (SDS) (Aldrich) 2.4. Activity measurements of enzyme electrodes
were used as received. Glucose oxidase (E.C. 1.13.4),
peroxidase (E.C., o-dianasidine, sulphuric acid The activities of free and immobilized invertase were
and hydrogen peroxide were obtained from Merck. determined according to Nelson method [18]. Different
MBTA was synthesized by esterification of thio- concentrations of sucrose solution were preincubated for
phene-3-acetic acid (Fluka) and (S)-(-)-2 methylbutanol 10 min at 25 °C. Then, enzyme electrode was placed into
(Aldrich) in the presence of p-toluene sulfonic acid, sucrose solutions for specific reaction times (2, 4, 6 min).
(PTSA) (Scheme 1). After removing the electrode, 1 ml of aliquots were
drawn and 1 ml of Nelson reagent was added to termi-
2.2. Electrochemical polymerization of MBTA nate the reaction. Then, tubes were placed in boiling
water for 20 min. After cooling, 1 ml arsenomolybdate
[CH3 (CH2 )3 ]4 NBF4 (0.65 g) and MBTA (50 mg) were was added as the coloring agent. Next, the solutions
dissolved in dichloromethane (10 ml). Polymerization were diluted to 7 ml with distilled water. Absorbances


+ OH
S benzene S

Scheme 1. Synthesis of MBTA.

S. Isßık et al. / European Polymer Journal 39 (2003) 2375–2381 2377

Polymerization N N

S n x S n y

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

for the blank and the solutions were measured at 540 nm bilization of invertase and glucose oxidase [5–7,20]. The
(a Shimadzu UV-1601 model spectrophotometer was activities of the invertase immobilized in SDS-system
used). For the free invertase activity determination, was found to be the best, however, for glucose oxidase
varying concentrations of sucrose solutions were pre- all the electrolytes showed almost the same activity in
incubated for 10 min at 25 °C and 0.1 ml of enzyme the presence of different supporting electrolytes. The
solution (0.01 mg/ml) was added. After the reaction for electrolysis times for immobilizations of invertase and
specific times, 1 ml Nelson reagent was added. Rest of glucose oxidase were different for different electrolytes;
the procedure was the same as in the case of invertase 30 min electrolysis with SDS, 120 min or more with both
activity determination. PTSA and NaPTS. Therefore, for the rest of the work,
The activity assay for free and immobilized glucose SDS was used as the supporting electrolyte for both
oxidase was carried out according to a modified version enzymes due to the minimum time requirement for the
of Sigma Bulletin [19]. For that purpose, different con- immobilization.
centrations of glucose solutions (2.5–50 mM) were pre- Because of using low concentration of supporting
pared (in buffer). Then, they were placed in water bath, electrolytes for the enzyme entrapment during the im-
shaken for 10 min at 25 °C for preincubation. Enzyme mobilization, conductivities of enzyme immobilized films
electrode was then placed in glucose solution and the were found to be in the order of 103 S/cm.
reaction was carried out for specific times. After re-
moving the electrode, 0.5 ml of aliquots were drawn and 3.2. Morphology of immobilized enzymes
0.1 ml POD (60 U/ml), 2.4 ml o-dianisidine (21 mM) as
the reducing agent were added. The reaction was stop- The morphologies of enzyme entrapped films were
ped with the addition of 0.5 ml sulfuric acid (2.5 M). investigated by scanning electron microscopy (SEM) by
After mixing, absorbances for blank and the solution JEOL JSM-6400. For the solution sides of the SDS
were measured at 530 nm. For the free enzyme, different doped PMBTA/PPy film without enzyme (Fig. 1a), the
concentrations of glucose solutions were prepared and cauliflower-like structure which was usually observed for
preincubated in water bath for 10 min at 25 °C. Then 0.1 copolymers of pyrrole was dominant. For the invertase
ml glucose oxidase added and reacted for specific times. (Fig. 1b) and glucose oxidase (Fig. 1c) entrapped films,
The rest of the procedure was the same as the enzyme the cauliflower-like structure was significantly damaged.
electrode case. Moreover, enzyme clusters were observed in the struc-
One unit of invertase activity was defined as amount ture. The type of supporting electrolyte is most crucial
of enzyme required to release 1 lmol glucose equivalent according to our previous studies [21,22]. Since the
per minute at pH 5.1, 25 °C. One unit of glucose oxi- anion of the electrolyte acts as the dopant ion for the
dase activity was defined as the oxidation of 1 lmol of b- conducting polymers/copolymers where the surface mi-
D -glucose to D -gluconic acid and H2 O2 per minute at
crostucture of the film is affected. This effect in return
pH 5.1, 25 °C. plays another role in the efficiency of the immobiliza-
tion. This is due to the lack of cauliflower morphology
on the film surface. The enzyme is not only embedded in
3. Results and discussion the polymer but also trapped on the surface which
makes them visible under SEM.
3.1. Effect of supporting electrolytes on immobilization
3.3. Effect of temperature on activity of enzymes
Different supporting electrolytes, p-toluene sulfonic
acid (PTSA), sodium p-toluene sulfonate (NaPTS) and The optimum temperatures for PPy/invertase (Fig.
sodium dodecyl sulfate (SDS) were used for the immo- 2a), PPy/PMBTA/invertase (Fig. 2b) matrices prepared
2378 S. Isßık et al. / European Polymer Journal 39 (2003) 2375–2381

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. Effect 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.
S. Isßık et al. / European Polymer Journal 39 (2003) 2375–2381 2379

in the presence of SDS were found as 50 °C. Invertase PPy electrode, because after reaching maximum activity
entrapped PMBTA/PPy electrode was more stable than 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 immobi- Table 2
lized invertase at 25 °C and at optimum pH Kinetic constant for glucose oxidation by free and immobilized
Km (mM) Vmax (lmol/min) glucose oxidase at 25 °C and at optimum pH

Free invertase 59 82.3a Km (mM) Vmax (lmol/min)

PPy/invertase 53 2.6b Free glucose oxidase 18.5 16.8a
PPy/PMBTA/in- 87 6.1b PPy/glucose oxidase 21.7 1.0b
vertase PMBTA/PPy/glucose oxidase 22 0.8b
a a
Per ml solution. Per ml solution.
b b
Per electrode. Per electrode.





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.
2380 S. Isßık et al. / European Polymer Journal 39 (2003) 2375–2381



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

optimum temperature for PPy/glucose oxidase (Fig. 2c), 3.5. Operational stability and shelf life of the enzyme
PPy/PMBTA/glucose oxidase electrodes prepared in electrodes
SDS was also found to be 30 °C (Fig. 2d). For free
glucose oxidase, optimum temperature of about 30 °C To estimate the stability of electrodes in terms of
was reported [20]. This shows the similarity of micro- repetitive uses, 20 successive measurements were per-
environments of the immobilized enzymes and the free formed at 25 °C during one day for both enzymes (Fig.
enzymes. 3). For the immobilized invertase in PPy/PMBTA ma-
trices (Fig. 3a) almost no change in their activity was
observed. For the immobilized glucose oxidase with the
3.4. Kinetic parameters of immobilized enzyme same matrix (Fig. 3c), a small change in the activity was
obtained. For the immobilized invertase in PPy matrices
The kinetic studies were performed for free and (Fig. 3b) small fluctuations were observed compared to
immobilized enzymes at different concentrations of PMBTA matrices. However, the immobilized glucose
substrate. The apparent Michaelis constant (Km ) and oxidase in PPy matrices (Fig. 3d), revealed same be-
maximum velocity ðVmax Þ were determined from Line- havior compared to the PPy/PMBTA matrices, in terms
weaver–Burk plots [23]. For the invertase entrapped in of activity and Km . Invertase entrapped in PPy/PMBTA
PPy and PMBTA/PPy matrices, there is an increase in matrices have storage stability (Fig. 4a) i.e., in 60 days
Km values compared to free invertase (Table 1). The only 7% of invertase lost its activity whereas in PPy
increase in Km values (decreased affinity) is due to matrices between 1 and 15 days it loses its activity to
electrostatic interactions between the substrate and 40% then between the 15 and 30 days its activity remains
matrix and also diffusion effects. Enzyme–substrate constant and thereafter it further loses activity (Fig. 4b).
complex formation becomes more difficult due to the Immobilization of glucose oxidase in these matrices has
structure of the conducting polymers (low porosity, no storage stability, almost after one day glucose oxi-
rigid structure, etc.) for immobilized enzymes. How- dase loses its activity.
ever, Km values for immobilized glucose oxidase were
comparable that of free enzyme (Table 2). It was
concluded that immobilization did not cause any hin- 4. Conclusions
drance of enzyme towards its substrate. The decrease in
the reaction rate ðVmax Þ of both immobilized invertase This work shows that PMBTA/PPy copolymer can be
and glucose oxidase may be attributed to the restricted used as enzyme immobilization matrix for invertase and
diffusion of substrate to the enzyme. For the immobi- glucose oxidase. SDS was found to be the best sup-
lized invertase, PMBTA/PPy matrices exhibited higher porting electrolyte for the entrapments. Both enzymes
reaction rates than PPy matrices. However, for the were dispersed homogeneously in the electrode since
immobilized glucose oxidase, reaction rate is almost the they show appreciable activity with respect to free en-
same for both matrices. zyme activity. Immobilization of invertase in PMBTA/
S. Isßık et al. / European Polymer Journal 39 (2003) 2375–2381 2381

PPy matrices is more feasible than in PPy matrices as [7] Alkan S, Toppare L, Yagcı Y, Hepuzer Y. J Biomater Sci
regards to their optimum temperature and storage sta- Polym Edn 1999;10:1233.
bility. Immobilization of glucose oxidase for both [8] Yasuzawa M, Niede T, Hirana T, Kunugi A. Sensors
PMBTA/PPy and PPy matrices reveal almost the same Actuat B: Chem 2000;66:77.
[9] Almeida NF, Beckman EJ, Ataai MM. Biotechnol Bioeng
stability. As to the stability, in PMBTA/PPy matrices,
invertase immobilization is more feasible than that of [10] Cosnier S, Lepellec A, Guidetti B, Rico-Lattes I. J Elec-
glucose oxidase. troanal Chem 1998;449:165.
[11] Diaz AF, Kanazawa KK, Gardini GP. J Chem Soc Chem
Commun 1979:635.
Acknowledgements [12] Monsan P, Combes O. Biotechnol Bioeng 1984;26:347.
[13] Akbulut U, Sungur S, Pekyardimci S. Macromol Rep
This work is partially supported by TUBITAK re- 1991;28:239.
search funds TBAG-2221 (102T116) and METU BAP- [14] Marek M, Valentova O, Kas J. Biotechnol Bioeng
[15] Mansfeld J, F€orster M, Schellenberger A, Dautzenberg H.
Enzyme Microb Technol 1991;13:240.
[16] Home PD, Alberti KGM. Biosensors: fundamentals and
References applications. Oxford: Oxford Press; 1987. p. 723.
[17] Levent A, Toppare L, Cianga I, Yagcı Y. Macromol Chem
[1] Mesing R. Immobilized enzymes for industrial reactors. Phys 2003;204:1118.
Academic Press; 1995. [18] Nelson N. J Biol Chem 1994;153:375.
[2] Chibata I. Immobilized enzymes. New York: Kodansha [19] The enzymatic colorimetric determination of glucose,
Ltd., Halsted Press; 1978. Sigma Technical Bulletin No 510, St Louis USA: Sigma
[3] Foulds NC, Lowe CR. J Chem Soc Faraday Trans Chemical Co.; 1983.
1986;82:1259. € nen A, Yagcı Y.
[20] Tirkesß S, Toppare L, Alkan S, Bakır U, O
[4] Bartlett PN, Whitaker RG. J Electroanal Chem 1987; Int J Biol Macromol 2002;30:81.
224:27. [21] Alkan S, Toppare L, Yagci Y, Hepuzer Y. J Biomater Sci
[5] Selampınar F, Akbulut U, O € zden MY, Toppare L. Bioma- Polym Ed 1999;10:1223.
terials 1997;18:1163. [22] Enginer R, Toppare L, Alkan S, Bakır U. React Funct
[6] Kızılyar N, O € zden MY, Toppare L, Yagci Y. Synth Met Polym 2000;45:227.
1999;104:45. [23] Lineweawer H, Burk D. J Am Chem Soc 1934;56:658.