European Polymer Journal 39 (2003) 2375–2381

www.elsevier.com/locate/europolj

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

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

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

Enzymes are protein molecules which serve to accel-
erate 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 pharma-
ceutical industries). Furthermore, the immobilized en-
zyme has a longer half-life and predictable decay rate [1].
Enzymes are immobilized by carrier binding (physical
adsorption, ionic binding, covalent bonding), by cross-
linking, adsorption or entrapment methods [2]. The en-
trapment of enzymes in conducting polymer matrices
during electrochemical polymerization is attracting great
*
Corresponding author. Tel.: +90-312-210-3251; fax: +90-
312-210-1280.
E-mail address: toppare@metu.edu.tr (L. Toppare).
1
On leave from ‘‘Petru Poni’’ Institute of Macromolecular
Chemistry, Iasi, Romania.
0014-3057/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0014-3057(03)00184-8
and inexpensive. Moreover, enzyme molecules can be
entrapped during electropolymerization in one step and
polymer film uniformly covers the surface of the sub-
strate electrode of any shape or size. Furthermore, the
film 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 [3–7]. In literature, several works were re-
ported on the immobilization of enzymes in polypyrrole
and its derivatives [8–11].
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 hydro-
lyze sucrose (to glucose and fructose) in the preparation
of invert sugar which has a lower crystallinity than su-
crose 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
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.
Glucose oxidase catalyzes the oxidation of glucose to
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
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 offers the possibility of continuously self-
monitoring 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 character-
ization of both PMBTA and PMBTA/PPy copolymer
was described in detail previously [17]. Optimum tem-
perature and kinetic parameters (Vmax , the maximum
reaction rate and Km the affinity of enzyme towards
substrate) for the immobilized enzymes were examined.
Furthermore, surface morphologies of enzyme en-
trapped films were investigated. Operational and storage
stabilities of the enzyme electrodes were determined.
2. Experimental
2.1. Materials
Tetrabutylammonium tetrafluoroborate, [CH3 -
(CH2 )3 ]4 NBF4 was obtained from Aldrich and dichlo-
romethane 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 esterification of thio-
phene-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
+ OH
S benzene
Scheme 1. Synthesis of MBTA.
Galvanostatic
O
O Polymerization
O O
S S n
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 elec-
trochemical polymerization of pyrrole either on bare or
PMBTA coated platinum electrodes. The electrolysis
solution consisted of invertase (1.0 mg ml
1 ), SDS (0.6
mg ml
1 ) as the supporting electrolyte and acetate buffer
(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
buffer 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 ml
1 ) (Scheme 3).
2.4. Activity measurements of enzyme electrodes
The activities of free and immobilized invertase were
determined according to Nelson method [18]. Different
concentrations of sucrose solution were preincubated for
10 min at 25 °C. Then, enzyme electrode was placed into
sucrose solutions for specific 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 termi-
nate 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
PTSA CO-O
S
MBTA
 S. Isßık et al. / European Polymer Journal 39 (2003) 2375–2381
H
N
O O
O O
Potentiostatic
Polymerization
S n
PMBTA
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 pre-
incubated for 10 min at 25 °C and 0.1 ml of enzyme
solution (0.01 mg/ml) was added. After the reaction for
specific 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 modified version
of Sigma Bulletin [19]. For that purpose, different con-
centrations of glucose solutions (2.5–50 mM) were pre-
pared (in buffer). 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 specific times. After re-
moving 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 stop-
ped 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, different
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 specific times.
The rest of the procedure was the same as the enzyme
electrode case.
One unit of invertase activity was defined as amount
of enzyme required to release 1 lmol glucose equivalent
per minute at pH 5.1, 25 °C. One unit of glucose oxi-
dase activity was defined as the oxidation of 1 lmol of b-
D -glucose to D -gluconic acid and H2 O2 per minute at
pH 5.1, 25 °C.
3. Results and discussion
3.1. Effect of supporting electrolytes on immobilization
Different supporting electrolytes, p-toluene sulfonic
acid (PTSA), sodium p-toluene sulfonate (NaPTS) and
sodium dodecyl sulfate (SDS) were used for the immo-
2377
H H
N N
x S n y
PMBTA/PPy
bilization of invertase and glucose oxidase [5–7,20]. 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 different supporting electrolytes. The
electrolysis times for immobilizations of invertase and
glucose oxidase were different for different 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 im-
mobilization, conductivities of enzyme immobilized films
were found to be in the order of 10
3 S/cm.
3.2. Morphology of immobilized enzymes
The morphologies of enzyme entrapped films were
investigated by scanning electron microscopy (SEM) by
JEOL JSM-6400. For the solution sides of the SDS
doped PMBTA/PPy film without enzyme (Fig. 1a), the
cauliflower-like structure which was usually observed for
copolymers of pyrrole was dominant. For the invertase
(Fig. 1b) and glucose oxidase (Fig. 1c) entrapped films,
the cauliflower-like structure was significantly damaged.
Moreover, enzyme clusters were observed in the struc-
ture. 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 mi-
crostucture of the film is affected. This effect in return
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
the polymer but also trapped on the surface which
makes them visible under SEM.
3.3. Effect of temperature on activity of enzymes
The optimum temperatures for PPy/invertase (Fig.
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.

(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.
2380 S. Isßık et al. / European Polymer Journal 39 (2003) 2375–2381
(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 micro-
environments of the immobilized enzymes and the free
enzymes.
3.4. Kinetic parameters of immobilized enzyme
The kinetic studies were performed for free and
immobilized enzymes at different concentrations of
substrate. The apparent Michaelis constant (Km ) and
maximum velocity ðVmax Þ were determined from Line-
weaver–Burk 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 affinity) is due to
electrostatic interactions between the substrate and
matrix and also diffusion effects. Enzyme–substrate
complex formation becomes more difficult due to the
structure of the conducting polymers (low porosity,
rigid structure, etc.) for immobilized enzymes. How-
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-
drance of enzyme towards its substrate. The decrease in
the reaction rate ðVmax Þ of both immobilized invertase
and glucose oxidase may be attributed to the restricted
diffusion of substrate to the enzyme. For the immobi-
lized 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.
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 per-
formed at 25 °C during one day for both enzymes (Fig.
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
same matrix (Fig. 3c), a small change in the activity was
obtained. For the immobilized invertase in PPy matrices
(Fig. 3b) small fluctuations were observed compared to
PMBTA matrices. However, the immobilized glucose
oxidase in PPy matrices (Fig. 3d), revealed same be-
havior 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 oxi-
dase loses its activity.
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 sup-
porting electrolyte for the entrapments. Both enzymes
were dispersed homogeneously in the electrode since
they show appreciable activity with respect to free en-
zyme activity. Immobilization of invertase in PMBTA/
 S. Isßık et al. / European Polymer Journal 39 (2003) 2375–2381
PPy matrices is more feasible than in PPy matrices as
regards to their optimum temperature and storage sta-
bility. 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 re-
search funds TBAG-2221 (102T116) and METU BAP-
2002-01-03-01.
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