Electrochemically synthesized polyaniline as support for lipase immobilization

Article abstract of DOI:10.1016/j.molcatb.2011.02.004

Electrochemical synthesis of polyaniline support for enzyme immobilization provides easier control over the properties of obtained polymer and reduced risk of biocatalyst inactivation with residues of toxic compounds. In the present study, immobilization

Full text of DOI:10.1016/j.molcatb.2011.02.004

Journal of Molecular Catalysis B: Enzymatic 70 (2011) 55–60
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Electrochemically synthesized polyaniline as support for lipase immobilization
a,∗
b
a
a
a
Dejan Bezbradica , Branimir Jugovi c´ , Milica Gvozdenovi c´ , Sonja Jakoveti c´ , Zorica Kne zˇ evi c´ -Jugovi c´
a
Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia
Institute of Technical Science, Serbian Academy of Science and Arts, Knez Mihajlova 35, 11000 Belgrade, Serbia
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Electrochemical synthesis of polyaniline support for enzyme immobilization provides easier control over
the properties of obtained polymer and reduced risk of biocatalyst inactivation with residues of toxic
compounds. In the present study, immobilization of lipase from Candida rugosa on electrochemically syn-
thesized PANI (activated with glutaraldehyde) resulted with high lipase loadings up to 93.7 mg of proteins
per gram of dry support. The activation of support and immobilization were optimized, with respect to
activity yield. The optimum concentration of glutaraldehyde was 2% (w/v) and optimum concentration
of enzyme was 4 mg ml . Modification of enzyme surface with carbodiimide and ethylenediamine was
performed in order to increase concentration of amino groups. Aminated lipase exhibited higher specific
activity (52%) and thermal stability (3 times) after immobilization, compared with non-modified lipase.
Also, reusability of immobilized enzyme was significantly increased with amination, especially if immo-
bilization was performed at pH 10, so in such a way obtained derivative retained 91% of activity after 15
reaction cycles.
Received 26 October 2010
Received in revised form 30 January 2011
Accepted 1 February 2011
Available online 25 February 2011
Keywords:
Lipase
Amination
Polyaniline
Electrochemical synthesis
Galvanostatic technique
−1
©
2011 Elsevier B.V. All rights reserved.
1
. Introduction
terol oxidase [15], lipase [7], and horseradish peroxidase [16] can
also be found.
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) gain huge
In last decade, PANI was recognized as excellent support for
enzyme immobilization due to its high physical and chemical
stability, large retention capacity, and good morphological charac-
teristics [17,18]. Prior to immobilization PANI is usually activated
using glutaraldehyde, in order to introduce carbonyl group and
facilitate covalent immobilization via amino groups of enzymes.
In such a way immobilized trypsin and horseradish peroxidase
exhibited good activity and increased stability at extreme pH val-
ues [17,19]. In a study with glucoamylase, the increase of specific
enzyme activity after immobilization was observed and immobi-
lized enzyme showed high reusability, thermal and storage stability
[20]. Important advantage of PANI-based supports is possibility of
preparation of nanofibre structures, which ensure reduced mass
transfer limitation, and simultaneously enables easier recovery
than nanoparticles or carbon nanotubes [21]. In a study with lipase
immobilized on polyaniline nanofibres by adsorption and sub-
sequent glutaraldehyde cross-linking high stereo-selectivity and
stability of immobilized enzyme was achieved. Also, it was demon-
strated that lipase immobilized on polyaniline nanofibres can be
easily separated by low-speed centrifugation [21] or even using
magnets if iron-oxide was applied in preparation of nanofibres [22].
The aim of this study was to investigate prospects of using
polyaniline obtained by electropolymerization as support for
immobilization of lipase from Candida rugosa. Electrochemical syn-
interest of researchers because they catalyze reactions at
hydrophobic/hydrophilic interfaces (such as hydrolysis and trans-
esterification of fats and oils, ester synthesis) usually with high
enantioselectivity [1–4]. A number of immobilization supports and
methods have been applied in lipase immobilization in order to
extend reusability and increase their stability towards high tem-
peratures and organic solvents [5,6].
Polyaniline (PANI) is a semi-flexible rod organic polymer, which
recently has drawn attention of researchers in area of biotechnol-
ogy. Due to its good electrical conductivity, low-cost monomers,
ease of synthesis, good mechanical strength, and simple doping
chemistry, PANI is very attractive polymer for constructing elec-
trochemical biosensors [7–9]. Numerous investigations of PANI
application in electrochemical biosensors has been reported, with
vast majority focused on glucose sensors with imobilized glu-
cose oxidase [9–12], but examples of PANI-based biosensors with
immobilized lactate dehydrogenase [13], tyrosinase [14], choles-
∗ Corresponding author at: Faculty of Technology and Metallurgy, Biochemical
Engineering and Biotechnology, University of Belgrade, Karnegijeva 4, 11000 Bel-
grade, Serbia. Tel.: +381 11 3303776; fax: +381 11 3370387.
E-mail address: dbez@tmf.bg.ac.rs (D. Bezbradica).
1
381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.02.004

5
6
D. Bezbradica et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 55–60
thesis using galvanostatic technique was reported to provide better
control over reaction rate and morphological properties of obtained
polymer and has proven to be acceptable for enzyme immobi-
lization [9,23,24]. Also, it is performed with significantly lower
amount of toxic compounds than ordinary organic synthesis, lead-
ing to reduced risk of enzyme inactivation during immobilization
by residues of these compounds. In this study, the immobilization
process was optimized with regard to glutaraldehyde concen-
tration during activation, pH and enzyme concentration during
immobilization, in order to promote covalent immobilization.
In a final stage of study, chemical modification of lipase with
2.2.3. Lipase immobilization on PANI powder
Then, activated support was transferred into flask with 10 ml of
C. rugosa lipase solution in 10 ml of sodium phosphate buffer for
◦
−1
16 h at 25 C in orbital shaker at 150 min . The concentration of
lipase was varied between 1 and 6 mg ml−1.
2.2.4. Chemical modification of lipase
Amination of suspended lipase was performed using 1 M solu-
−2
−3
tion of EDA with 10 or 10 M of EDAC at pH 4.75. After 90 min
◦
of gently stirring at 25 C, the modified preparations were filtered
and incubated for 4 h with a 0.1 M hydroxylamine solution at pH 7
1
-ethyl-3-(dimethylamino-propyl) carbodiimide (EDAC) and ethy-
◦
and 4 C to recover EDAC-modified tyrosines [25].
lene diamine (EDA) was applied in order to introduce additional
amino groups, promote multipoint covalent attachment of enzyme,
and improve stability of immobilized enzyme.
2
.2.5. The protein assay
The protein concentration was determined spectrophotomet-
rically at ꢀ = 595 nm (Ultrospec 330 pro, Amersham Biosciences)
using standard Bradford assay [26].
2
. Experimental
2
.2.6. Activity assay
Lipase activity was determined by method based on measur-
2.1. Materials
ing the effects of the hydrolysis of p-nitrophenyl palmitate (pNPP).
The absorbance was measured at 410 nm against substrate free
blank. The standard graph was prepared by using p-nitrophenol.
One international unit (IU) is defined as the amount of enzyme that
liberated 1 ␮mol p-nitrophenol per min under the assay conditions.
Lipase from C. rugosa, glutaraldehyde (25% solution), EDAC,
hydroxylamine, and EDA were purchased from Sigma (St. Louis,
MO, USA). Aniline and Coomassie protein assay reagent were pur-
chased from Fluka (Buchs, Switzerland). For polishing electrode,
polishing alumina (1 ␮m, Banner Scientific Ltd., Coventry, UK)
and polishing cloths (Buehler Ltd., Lake Bluff, IL, USA) were used.
p-Nitrophenyl palmitate was purchased from Alfa Aesar Gmbh
3. 3.Results and discussion
(
Karsluhe, Germany). Other reagents and solvents used are ana-
3.1. PANI powder morphology
lytical grade.
After electrochemical synthesis, polyaniline was mechanically
detached from graphite electrode and microscopic analysis of
morphology and size of obtained PANI powder was performed.
Obtained microphotographs (Fig. 1) show that electrochemical syn-
thesis of PANI leads to formation of particles of non-uniform shape
and size. It can be noticed that smallest particles are prevalently
rod-shaped particles with size of several ␮m. Agglomeration leads
to formation of irregular, drastically larger, conglomerate particles,
with largest diameter of around 200 ␮m. On large conglomerates,
pores with dimensions around 1 ␮m are noticeable (Fig. 1b), and
within such pores lipase could also be readily immobilized, since C.
rugosa lipase dimensions are approx. 7 nm × 5 nm × 7 nm [27].
2
2
.2. Methods
.2.1. Electrochemical synthesis of polyaniline
Electrochemical polymerization of PANI on graphite electrode
was performed galvanostatically at constant current density of
j = 2.0 mA cm from aqueous solution of 1.0 mol dm HCl contain-
ing 0.25 mol dm aniline on both sides of the graphite electrode
−
2
−3
−3
2
(
S = 100 cm ) inserted between two stainless steel plates, serving as
2
counter electrodes (S = 50 cm ). Prior to electrochemical synthesis,
graphite electrode was first mechanically polished with fine emery
papers (2/0, 3/0 and 4/0 respectively) and then with polishing alu-
mina on polishing cloths, the traces of the polishing alumina were
ultrasonically removed from the electrode surface during 5 min.
Prior to use, aniline was distilled in argon atmosphere. Polymeriza-
tion was performed in the prismatic polyethylene electrochemical
reactor with a volume of 0.8 dm . After synthesis, polyaniline pow-
der was mechanically detached from elctrode. PANI powder was
rinsed repeatedly with distilled water to the negative reaction on
chloride ions, and dried at 90 C under vacuum. When the water
insoluble PANI mass was determined, acetone soluble olygomers
were removed in Soxhlet extractor during 3 h, than washed with
distilled water and dried in the vacuum at 90 C. Micrograph of the
PANI deposit on graphite electrode was taken by optical microscope
3.2. The immobilization of lipase from C. rugosa
The initial part of our study was focused on optimization of
key factors of support activation and lipase immobilization. PANI
particles were modified previous to immobilization using GA, in
order to introduce reactive carbonyl groups. Low concentration of
introduced GA residues can lead to insufficient immobilization of
enzyme, while conversely high concentration can lead to unwanted
cross-linking of enzyme and distortion of catalytic conformation
[28,29]. Therefore, the concentration of GA during activation of
support was optimized with respect to obtained enzyme load and
hydrolytic activity of obtained immobilized lipase. Immobiliza-
3
◦
◦
(
Olympus CX41, Camera Olympus UC30), interfaced to PC.
−
1
tion was performed with 6 mg ml in immobilization mixture. All
experiments have been carried out in triplicate, so average values
and standard errors are illustrated inFig. 2.
2
.2.2. Activation of PANI powder
The activation of support was performed by incubation of 0.5 g
Obtained results show that variation of GA concentration
between 1 and 5% insignificantly influenced concentration of lipase
immobilized on support, resulting with concentrations in very nar-
of polyaniline powder in 20 ml of glutaraldehyde (GA) solution in
◦
2
0 mM sodium phosphate buffer (pH 7,0) for 1 h at 25 C in orbital
−1
−1
shaker at 150 min . The concentration of GA was varied within
range 1–5% (v/v). After incubation, activated polyaniline support
was filtered and thoroughly rinsed in order to elute unbounded GA
and avoid unwanted cross-linking.
row range between 88.3 and 93.7 mg g of dry support. On the
other hand, the effect on activity of immobilized lipase was very
significant. The highest activity of immobilized lipase was achieved
−
1
when support was activated with 2% solution of GA (7.5 IU g of

D. Bezbradica et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 55–60
57
Fig. 1. Micrographs of the PANI powder obtained by electrochemical synthesis.
support), and further increase of GA concentration led to steep
decrease of activity.
investigation of GA concentration effect on activation process was
undertaken by Betancor et al. and conditions allowing formation of
monolayer or bilayer of glutaraldehyde on support surface were
established [28]. The activation time applied in our study was
adequate for mild activation leading to formation of monolayer
of GA on support. Reduced activity of immobilized lipase, which
occurs at GA concentration above 2%, can be ascribed to result-
ing dense distribution of GA residues on surface of support and
increased probability of cross-linking leading to disturbance of
lipase catalytic active conformation. Almost insignificant effect of
GA concentration on concentration of immobilized enzyme indi-
cates that applied concentration of lipase was high enough to reach
saturation at observed GA concentration range.
The chemistry of glutaraldehyde activation is not completely
elucidated, since a variety of glutataldehyde structures can be
formed at usual conditions of support activation, such as its mono-
and dihydrates, as well as cyclic hemiacetal and oligomers [30].
Despite ambiguities in elucidation of the bond formation mech-
anism, GA activation has been successfully applied for long time
and the effects of the most important process parameters on acti-
vation of support have been previously analyzed [29]. Thorough
The effect of offered lipase amount was investigated by varying
−
1
lipase concentration in solution in range 1–6 mg ml in immobi-
lization on PANI activated with 2% of GA. Experiments have been
carried out in triplicate, and results are illustrated in Fig. 2b. It is
obvious that the increase of offered enzyme led to more or less lin-
−
1
ear increase of enzyme load on support, reaching 91.2 mg g of
support. On the other hand, the activity of obtained derivatives
−
1
was increasing rapidly until around 60 mg g
was immobilized
and further increase of enzyme load cause only slight change of the
immobilized activity. Therefore, the specific activity (calculated per
−
1
mg of immobilized protein) reached highest values (98 IU mg
)
−
1
when immobilization was performed with 4 mg ml of enzyme
preparation in immobilization solution.
Obtained results of enzyme load (almost 100 mg g−1) indicate
that electrochemically synthesized PANI support has high bind-
ing capacity. Nevertheless, it seems binding allows retention of
catalytic conformation of lipase only in limited range of enzyme
concentration, since binding of additional amount of enzyme left
overall activity unchanged and specific activity reduced. It is plau-
sible that, when immobilization is performed at low concentration
of offered lipase, binding can occur with retaining catalytic confor-
mation of enzyme and at positions on surface of support which
allows easy access of support to active site of enzyme. In con-
trast, with higher concentration of offered enzyme in late stage
of immobilization process lipase binding probably occurs in less
favorable regions of support, maybe even in larger pores of PANI
conglomerates (Fig. 1b), which can hinder diffusion of substrate
during reactions.
3.3. Lipase amination and immobilization of modified lipase
In order to promote covalent immobilization and increase sta-
bility of immobilized lipase, enrichment of enzyme surface with
amino groups through chemical amination of carboxylic groups
of aspartic and glutamic side chains was performed. In Fig. 3 dis-
Fig. 2. The effect of (a) glutaraldehyde concentration during support activation and
(
b) initial lipase concentration in immobilization mixture on amount of immobilized
enzyme (ꢀ) and activity of immobilized enzyme (᭹).

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D. Bezbradica et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 55–60
Fig. 4. The effect of amination, applied EDAC concentration, and immobilization pH
on: (a) overall activity of immobilized lipase, and (b) specific activity of immobilized
lipase. Immobilization pH was 7 (dark gray bars) or 10 (light gray bars).
immobilization procedure. It is plausible that only slight impact
of chemical modification on activity of enzyme can be ascribed to
lower density of side chains with carboxylic groups in vicinity of
lid that covers catalytic triad (Fig. 3), compared with structures of
previously reported aminated lipases [31,32].
Both, modified and non-modified lipases, were immobilized at
pH 7 or pH 10. Immobilization at pH 10 was tested because pK
of ε-amino groups of lysine is around 10, hence in basic condi-
tions portion of non-dissociated groups is larger and this form is
more prone to react with carbonyl groups [34]. Immobilization
was performed with PANI support, activated with 2% GA, and in
all experiments immobilization mixtures of equal lipase activities
were applied. All experiments were carried out in triplicate and
obtained average activities of immobilized derivatives are illus-
Fig. 3. Distribution of Lys, Asp and Glu residues on the surface of lipase from C.
rugosa. Lys are shown in blue, Glu and Asp are shown in orange, and lid covering
active site is shown in pink. (a) Front view and (b) 180 rotation of the front view,
in x–y plane. The3D structure was obtained using Pymol vs. 0.99 and data obtained
from Protein Data Bank (PDB). PDB code of C. rugosa lipase: 1TRH. (For interpretation
of the references to color in this figure legend, the reader is referred to the web
version of this article.)
◦
−
1
trated in Fig. 4a. The highest activity (7.1 IU g ) was achieved
using non-modified lipase in immobilization performed at pH 7.
Generally, it can be observed that, in experiments at pH 7, activi-
ties of immobilized lipases continuously decrease with increasing
extent of modification, resulting with the lowest activity with
lipase modified with 0.01 M of EDAC. At pH 10, immobilized activ-
tribution of lysines, aspartic and glutamic acids on surface of C.
rugosa is illustrated. It can be observed that density of carboxylic
groups is significantly higher than amino groups, which implies
that amination of carboxylic groups can lead to drastic increase of
amino groups density enabling multipoint covalents attachement.
It was previously reported that chemical amination with EDA, after
activation with EDAC, has been successfully applied with several
industrially important lipases (lipases from T. lanuginosa, B. ther-
mocatenulatus and P. fluorescens), after adsorbing it on hydrophobic
support in order to protect undesired chemical changes in vicinity
of lipase active site and activity loss [31–33].
In our study, prior to immobilization the effects of chemical
amination of free suspended enzyme on retained activity were ana-
lyzed. The amination was performed with different concentrations
of EDAC (0.01 M or 0.001 M), which has been previously reported
to ensure complete or partial amination, respectively. It was found
out that lipase activity was not drastically reduced (26% and 14%,
respectively), so in following experiments amination was per-
formed without previous adsorption of lipase in order to simplify
−
1
ity of non-modified lipase is significantly reduced, to 4.8 IU g
.
Conversely, alkaline conditions were adequate for immobilization
of aminated lipases since only slight change of activities were
achieved. Besides activities, concentration of immobilized pro-
teins was also determined and used for calculation of specific
activities, which are presented in Fig. 4b. The highest specific
−
1
activity (93.7 IU g ) was measured for derivative obtained with
lipase modified with 0.01 M EDAC immobilized at pH 10. Generally,
enzyme loading was significantly lower when immobilization had
been performed at pH 10, resulting with higher specific activities
of these derivatives and different trend than in Fig. 4a. Also, lipase
amination exhibited favorable effects on specific activity, since the
increase of specific activity was observed with increase of applied
EDAC concentrations.

D. Bezbradica et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 55–60
59
Table 1
a
Half-lives (t1/2) of different C. rugosa lipase preparations determined in stability
assay at 60 C.
◦
Lipase
[EDAC] in amination, M
–
t1/2, h
5.3 ± 0.8
Immobilized at pH 7
0
0
–
0
0
–
.001
.01
5.7 ± 0.4
5.9 ± 0.5
6.9 ± 0.7
13.1 ± 1.1
15.9 ± 0.9
1.2 ± 0.4
0.9 ± 0.4
0.8 ± 0.3
Immobilized at pH 10
Free lipase
.001
.01
0.001
.01
0
The trend observed with derivatives obtained at pH 7 can be
explained by prevalence of adsorption mechanism due to high
concentration of charged lysine amino groups. The decrease of
immobilized activity and enzyme loading with enzyme modifica-
tion (Fig. 4a) can be ascribed to lower content of negatively charged
residues on enzyme surface, which reduces adsorption of enzyme
by electrostatic interactions with secondary amino groups on sur-
face of polyaniline. On the other hand, at pH 10 amino groups
are uncharged and covalent immobilization via carbonyl groups
can readily occur. Therefore, immobilized activity increased after
modification due to increased number of reactive amino groups
on enzyme surface. Specific activities were significantly higher for
derivatives obtained at pH 10. Since there is higher probability
for bond formation via area containing highest density of reactive
amino groups it is plausible immobilization occurs through area at
rear part of molecule (Fig. 4b), with respect to active site of lipase.
Therefore, observed increase of activity in immobilization at pH
b
1
0 could be due to more favorable orientation of enzyme during
immobilization process resulting with immobilized enzyme with
well-exposed active site, and consequently higher activity.
3.4. Thermal stability study
For better evaluation of immobilization support and methods
Fig. 5. Thermal stability assay: (a) the effect of immobilization pH. (ꢀ) lipase immo-
bilized at pH 7; (ꢁ) lipase immobilized at pH 10; (ꢁ) immobilization at pH 7 using
lipase aminated with 0.01 M EDAC; (᭹) immobilization at pH 10 using lipase ami-
nated with 0.01 M EDAC and (b) the effect of amination. The immobilization was
performed at pH 10 using: (᭹) non-aminated lipase; (ꢁ) lipase aminated with
it is necessary to compare stability of different enzyme deriva-
◦
tives. In our study, thermal stability assay was performed at 60 C
and pH 7. For easier comparison of stabilities half-lives of different
derivatives were determined, and obtained results are presented
in Table 1. The most illustrative results are depicted in Fig. 5.
It seems that both, pH of immobilization and chemical modifica-
tion of lipase exhibited strong influence on stability of immobilized
lipase. The immobilization at pH 10 resulted with derivatives of
significantly higher stability (Fig. 5a). For non-modified lipase,
immobilization at pH 10 led to increase of derivative half-life to
t1/2 = 6.9 h from t1/2 = 5.3 h obtained at pH 7, while for lipase modi-
fied with 0.01 M EDAC the increase was even larger-from t1/2 = 5.9 h
to t1/2 = 15.9 h. Lipase amination also has shawn clearly positive
effect on stability of immobilized derivatives, particularly in immo-
bilizations performed at pH 10 (Fig. 5b). Derivatives obtained using
aminated lipase exhibited around two-times higher half-lives than
derivatives obtained with non-modified lipases. Additionally, it is
noticeable that amination at higher concentration of EDAC (0.01 M)
resulted with significantly more stable derivative. These results
indicate that approach that combines introduction of additional
amino groups on lipase surface and immobilization at pH condi-
tions, enabled making of lipase derivative with stability three-times
higher in comparison with derivative obtained using conventional
approach. Also, aminated lipase immobilized at pH 10 exhibited
0.001 M EDAC; (ꢂ) lipase aminated with 0.01 M EDAC.
with carbonyl groups of activated PANI support have been formed
leading to significant increase of stability of immobilized lipase.
3.5. Reusability study
One of the most important advantages of immobilized enzymes
is the possibility of repeated use through a number of reaction
cycles, hence the prospect of recovering lipase immobilized on
PANI supports from reaction mixture and repeated use of deriva-
tives were analyzed. Immobilized derivative was separated using
low-speed centrifugation (4000 × g) after each reaction cycle and
re-suspended in fresh substrate. The effects of different strategies
for lipase immobilization on reusability were tested by comparison
of two derivatives: one prepared with non-modified lipase at pH 7
and other prepared with aminated lipase at pH 10 (Fig. 6).
Obtained results indicate that approach based on the increase of
number of reactive amino groups on lipase surface and immobiliza-
tion at basic conditions causes significant extension of immobilized
enzyme reuse. Namely, in such a way obtained derivative retained
91% of initial activity after 15 reaction cycles, while derivative
obtained using conventional approach (non-modified enzyme and
immobilization at pH 7) retained 73% of initial activity. These
1
6 times higher half-life than free lipase. Such a trend could also
be explained with previously stated assumption that immobiliza-
tion occurs via region containing higher density of amino groups.
As a consequence, it seems that higher number of covalent bonds

6
0
D. Bezbradica et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 55–60
thermal stability of immobilized enzyme for several times. Addi-
tionally, this strategy enabled important increase of operation
stability resulting with immobilized lipase that retains 91% of initial
activity after 15 reaction cycles. Even higher operational stability
was observed with enzyme immobilized on PANI-coated electrode
implying that applied combination of support preparation, enzyme
pre-modification, and immobilization conditions leads to highly
stable immobilized enzyme with good prospect in biosensor con-
struction.
Acknowledgment
The authors are grateful for financial support from Serbian Min-
istry of Science (project III 46010).
Fig. 6. The effect of lipase modification on reusability of PANI-immobilized lipases
in hydrolysis of p-NPP.
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2
[
[
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1
5 cycles. Results of this part of our study indicate that electro-
9
chemically synthesized PANI offers prospect of immobilization of
enzymes directly on electrode with similar activity retention as
in case of immobilization on PANI powder. Additionally, enzyme
immobilized on PANI-coated electrode exhibited even higher usage
stability than enzyme immobilized on PANI powder, which indi-
cates that this material should be considered in development of
biosensors.
[
9
[
[
[
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thesized PANI can be applied as immobilization support after
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−1
ities reaching 98 IU mg . Dimensions and morphology of obtained
PANI powder allows simple separation of immobilized derivative
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ity of obtained immobilized lipases was significantly improved
using approach which combines chemical modification of enzyme
prior to immobilization and optimization of immobilization pH.
Enrichment of lipase surface with amino group and performing
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