Highly stabilized lipase in polyaniline nanofibers for surfactant-mediated esterification of ibuprofen

Article abstract of DOI:10.1021/la404189e

Lipase (LP) from Candida rugosa was immobilized and stabilized in polyaniline nanofibers (PANFs) via a three-step process of enzyme adsorption, precipitation, and cross-linking, which generates the final immobilization called "EAPC". The activity of EAPC

Full text of DOI:10.1021/la404189e

Article
pubs.acs.org/Langmuir
Highly Stabilized Lipase in Polyaniline Nanofibers for Surfactant-
Mediated Esterification of Ibuprofen
Sung-Gil Hong, Han Sol Kim, and Jungbae Kim*
Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Republic of Korea
*
S Supporting Information
ABSTRACT: Lipase (LP) from Candida rugosa was immo-
bilized and stabilized in polyaniline nanofibers (PANFs) via a
three-step process of enzyme adsorption, precipitation, and
cross-linking, which generates the final immobilization called
“
EAPC”. The activity of EAPC was 5.1 and 5.9 times higher
than those of LP immobilizations via enzyme adsorption (EA)
and enzyme adsorption/cross-linking (EAC), respectively.
After incubation in an aqueous buffer under shaking (200
rpm) for 84 days, EAPC maintained 74% of its initial activity,
while EA and EAC retained 11 and 24% of their initial
activities, respectively. Highly stable and active EAPC was
employed for the resolution of racemic ibuprofen via esterification of S-(+)-ibuprofen with 1-propanol in isooctane. The addition
of 100 mM dioctyl sulfosuccinate (AOT) into the reaction medium increased the esterification activity by 61-fold, which can be
explained by the better dispersion of EAPC in isooctane. EAPC showed 42% conversion in the esterification of racemic ibuprofen
after 102 h, whereas EA and EAC showed only 1.2 and 1.4% conversion in the same condition, respectively. The EAPC approach
increases both loading and stability of LP, and the combination of EAPC with the surfactant addition can be employed for
efficient enzymatic reactions in organic solvents.
INTRODUCTION
have been tested as supporting materials for enzyme
■
2
,11
immobilization.
Polyaniline nanofibers (PANFs), in partic-
Because of the growing demand for environmentally friendly
catalysts working at ambient condition, a lot of interest and
efforts have been devoted to the use of enzymes. Enzymes are
eco-friendly biocatalysts that can catalyze chemical trans-
formations under mild reaction conditions, such as ambient
ular, can be used for the immobilization of enzymes in the form
12
1
of enzyme coatings and also have interfiber matrices that can
provide porous inner spaces for enzyme immobilization.
PANFs can be synthesized in a large scale more easily than
the other nanostructured materials, such as nanoparticles,
carbon nanotubes, mesoporous materials, and electrospun
nanofibers. Because of their structural and synthetic advantages,
2
−4
temperature, pressure, and neutral pH. Enzymes function in
various reaction media, such as water, organic solvents, and
5
,6
ionic liquids. From a biotechnological viewpoint, conducting
enzymatic reactions in organic solvents provides many
advantages, including suppression of water-dependent side
reactions, allowance of various synthetic reactions, and high
13−15
PANFs have been used to immobilize various enzymes.
Especially, the approach of enzyme adsorption, precipitation,
and cross-linking (EAPC), generating EAPC immobilization,
15
7
,8
could achieve high loading and stability of glucose oxidase.
solubility of organic compounds in non-aqueous solvents.
In the present work, we employed the EAPC method to
immobilize LP from Candida rugosa in PANFs and checked the
activity and stability of EAPC together with three control
samples: free LP, enzyme adsorption (EA), and enzyme
adsorption and cross-linking (EAC) methods. As a potential
application, the enzymatic esterification of racemic ibuprofen
with 1-propanol was performed in isooctane using EA, EAC,
and EAPC. One of the serious problems is the generation of
water from the esterification, which induces the aggregation of
Enzyme-catalyzed synthetic reactions, such as esterification,
transesterification, and aminolysis, have been performed in
organic solvents. In particular, interest in lipases (LPs), used as
9
biocatalysts in various industries, such as food, pharmaceutical,
10
detergent, and cosmetics, has been increasing because of their
esterification or transesterification reactions in organic solvents.
Despite these advantages, however, industrial applications of
most enzymes remain controversial because of their poor
9
stability. Repetitive usage of enzymes, potentially a major
1
6
immobilized LP. To solve this problem, we added a
surfactant, dioctyl sulfosuccinate (AOT), into the reaction
economic concern, is hampered by poor enzyme stability and
difficult enzyme recovery. As a potential solution, the
immobilization of enzymes on nanostructured materials has
been proposed to stabilize the enzyme activity and allow for
repeated enzyme usage. With the recent development of
nanobiocatalytic approaches, various nanostructured materials
Received: October 30, 2013
Revised: January 3, 2014
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/la404189e | Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Figure 1. SEM images of (a) pristine PANFs, (b) EA, (C) EAC, and (d) EAPC.
medium potentially to prevent or alleviate the aggregation of
immobilized LP in PANFs over the course of enzymatic
esterification in isooctane.
a control. Tris-HCl buffer (100 mM, pH 7.0) for the capping of
unreacted aldehyde groups was replaced to PB buffer (100 mM, pH
7
.0) to prevent the addition of nitrogen into the samples of EAC and
EAPC. Samples were dried via freeze-drying after washing by distilled
water excessively. Then, the samples were analyzed by an elemental
analyzer (Flash 2000, Thermo Scientific, Waltham, MA).
EXPERIMENTAL SECTION
■
Materials. LP from C. rugosa, 4-nitrophenyl butyrate (4-NB),
glutaraldehyde (GA), ammonium sulfate, ammonium persulfate,
hydrochloric acid (HCl), (±)-2-(4-isobutylphenyl) propanoic acid
ibuprofen), sodium phosphate, methyl heptadecanoate, and 2-amino-
-hydroxymethyl-propane-1,3-diol (Tris) buffer salts were purchased
Activity and Stability Measurements. The activity was
measured by the hydrolysis of 4-NB in 10 mM PB (pH 7.0). The
enzyme samples were mixed with 50 mM substrate solution, and the
increase of absorbance at 410 nm was measured using a
spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan). The initial
rate of each sample was calculated from the time-dependent increase
of absorbance at 410 nm within 10% conversion. The stability of
enzyme samples was checked by measuring the activity time-
dependently after incubation of each sample in 10 mM PB (pH 7.0)
under shaking (200 rpm) at both room temperature and 40 °C.
Esterification of an Ibuprofen Racemic Mixture and
Detection of Ibuprofen Ester by Gas Chromatography (GC).
EA, EAC, and EAPC were synthesized using 10 mg of PANF with 5
mL of LP solution (20 mg/mL) for the esterification of a (R,S)-
ibuprofen racemic mixture, and samples were lyophilized overnight
after washing with distilled water. Lyophilized samples were mixed
with isooctane containing 0−500 mM AOT. Then, 10 mM ibuprofen
racemic mixture, 20 mM 1-propanol, and 1% (v/v) distilled water were
added to the mixture. After gentle sonication, samples were shaken at
200 rpm at 40 °C. The mixtures were taken in a time-dependent
manner and mixed with additional isooctane-containing 40 mM
methyl heptadecanoate as an internal standard. The recycling
experiment was performed with 200 rpm shaking at 40 °C. Samples
were centrifuged and washed with isooctane every 4 h, and the same
amounts of racemic ibuprofen, water, and 1-propanol were then added.
All sample aliquots were analyzed by GC equipped with a flame
ionization detector (FID). To analyze the degree of esterification,
analysis of each aliquot was performed in an Agilent 7890 gas
chromatograph equipped with a FID and DB-5 column (30 m × 0.25
mm × 0.25 μm). The temperature of the injector, detector, and oven
were 300, 350, and 120 °C, respectively. The injection volume was 1
μL, and the column flow rate was 1 mL/min. The oven temperature
was increased at a rate of 20 °C/min from 120 to 250 °C with 3 min of
holding time. A typical GC chromatogram for the ibuprofen resolution
is shown in Figure S1 of the Supporting Information.
(
2
from Sigma (St. Louis, MO). AOT, aniline, 1-propanol, and 2,2,4-
trimethylpentane (isooctane) were purchased from Aldrich (Milwau-
kee, WI).
Preparation of PANFs. PANFs were synthesized by a mixture of
1
7
aniline and ammonium persulfate as an initiator in acidic conditions.
First, a mixture of 1 M HCl and aniline (volume ratio of 8.5:1.5) was
prepared, and the same volume of 0.1 M ammonium persulfate
solution in 1 M HCl was added to initiate the polymerization reaction
at room temperature under shaking (200 rpm) for 24 h. After
polymerization, PANFs were washed by distilled water 3 times. Finally,
the mass of PANFs was determined after vacuum drying at 50 °C for
1
7 h.
Immobilization of LP in PANFs. LP was immobilized in PANFs
by three different approaches: EA, EAC, and EAPC. A total of 20 mg/
mL LP solution in 100 mM sodium phosphate buffer (PB, pH 7.0)
was centrifuged at 10 000 rpm for 10 min and filtered by a syringe
(
0.22 μm pore size) to eliminate insoluble impurities. LP solution (1
mL) was mixed with PANFs (2 mg), and the mixture was shaken at
50 rpm at room temperature for 2 h, after which ammonium sulfate
solution was added to EAPC to a concentration of 55% (w/v). After
0 min of shaking at 150 rpm at room temperature, GA solution was
1
3
added to the EAPC sample to a final concentration of 0.5% (w/v). For
EA, ammonium sulfate and GA solution were replaced by 100 mM PB
(
1
pH 7.0), while in the case of EAC, ammonium sulfate was replaced by
00 mM PB (pH 7.0). For the immobilization, mixtures were
incubated on a tilt shaker (50 rpm) at 4 °C for 17 h. To cap unreacted
aldehyde groups, mixtures were shaken at 250 rpm with 100 mM Tris-
HCl buffer (pH 7.0) for 30 min after washing. In addition, LP-
immobilized PANF samples were washed 3 times under rigorous
conditions (250 rpm side-by-side shaking), followed by centrifugation
at 10 000 rpm. Lastly, EA, EAC, and EAPC were stored in 10 mM PB
(
pH 7.0) at 4 °C.
RESULTS AND DISCUSSION
■
Elemental analysis was performed to estimate the LP loadings of
EA, EAC, and EAPC in PANFs. The measured nitrogen contents were
used for the calculation of enzyme loadings, while PANFs were used as
Immobilization of LP from C. rugosa in PANFs. PANFs,
as a supporting material, have a porous interfiber matrix made
B
dx.doi.org/10.1021/la404189e | Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
by a complicated nanofiber network. LPs were immobilized in
PANFs using three different approaches: EA, EAC, and EAPC.
EA was prepared through simple enzyme adsorption, while
enzyme cross-linking by GA was added in the EAC approach.
EAPC was prepared by GA cross-linking of precipitated
enzymes after ammonium sulfate precipitation.
Figure 1 shows the scanning electron microscopy (SEM)
images of PANFs, EA, EAC, and EAPC. EA and EAC did not
cause significant changes in nanofiber thickness when
compared to pristine PANFs. On the other hand, EAPC
showed more thickly coated PANFs than EA and EAC. The
average nanofiber thicknesses of PANF, EA, EAC, and EAPC
were 74 ± 12, 98 ± 17, 114 ± 19, and 172 ± 28 nm,
respectively. A thick coating of EAPC can be explained by a
large amount of immobilized LPs in PANFs. In the case of
EAPC, LPs were aggregated and precipitated through a “salting-
out” effect. An increase in the ionic strength by ammonium
sulfate induces the aggregation and precipitation of LPs in
PANFs, allowing for more efficient cross-linking of LP
molecules upon GA treatment.
Figure 3. Stabilities of free LP, EA, EAC, and EAPC in aqueous buffer
at room temperature under shaking (200 rpm).
1
8
initial activity within 10 days. With 200 rpm of shaking at 40 °C
for 25 days, EAPC retained 82% of initial activity, whereas EA
and EAC retained 26 and 33% of initial activity, respectively
The LP loadings of EA, EAC, and EAPC, estimated by
measuring the nitrogen content of each sample via elemental
analysis, were 0.08, 0.12, and 0.62 mg of LP/mg of PANFs,
respectively (see Table S1 of the Supporting Information).
Interestingly, the enzyme loading goes well with the coating
thickness (see Table S2 of the Supporting Information).
Activity and Stability of EA, EAC, and EAPC in
Aqueous Solution. The activities per unit weight of PANFs
(
see Figure S2 of the Supporting Information).
The poor stability of EA and EAC is the result of continuous
denaturation and leaching of LPs from the PANFs, while the
enhanced stability of EAPC is closely related to the tight
packing and cross-linking of LP molecules on the PANFs. The
adjacent intermolecular distance between LP molecules,
induced by ammonium sulfate precipitation, has an important
effect on immobilization and stabilization, making cross-linkage
formation between LP molecules much easier. It is generally
accepted that a greater number of multi-point covalent linkages
on the enzyme surface would help to prevent the denaturation
−1
of EA, EAC, and EAPC were 0.83, 0.74, and 4.2 mM min
−1
mg of PANFs, respectively (Figure 2). The higher activity per
19
of enzyme molecules. The LP molecules of EAPC, cross-
linked with a greater number of multi-point linkages upon GA
treatment, would be more resistant to the denaturation that
leads the stabilized enzyme activity in the form of EAPC.
AOT-Mediated Esterification of (R,S)-Ibuprofen. LP
from C. rugosa can selectively esterify S-(+)-ibuprofen, which is
medicinally 100 times more active than R-(−)-ibuprofen, to S-
(
+)-ibuprofen ester with alcohol and water in organic
20−23
solvent.
For enzymes to be active in organic solvent, a
5
minimal amount of water is required. Water molecules
localized on the surface of the enzyme maintain its structural
flexibility in the organic solvent. The maximum rate of
enzymatic esterification was reached with a certain amount of
5,9
24
Figure 2. Activities per unit weight of PANFs (white bars) and LPs
water. However, the continuous production of water as a
(
gray bars) of EA, EAC, and EAPC.
result of the esterification reaction can be a problem in
16
hydrophobic organic solvents, such as isooctane. Water
molecules resulting from the esterification reaction can adsorb
on the hydrophilic surface of enzyme. Thus, continuous water
production can cause a serious aggregation of enzymes or
enzyme-immobilized nanostructured materials because of the
immiscible property between water and hydrophobic organic
solvents. It means that serious mass-transfer limitation of
substrates from the reaction medium to the active site of
enzyme can be caused during the reaction and, thus, reaction
efficiency can be dropped rapidly.
In this work, we varied the concentration of AOT, which can
induce better dispersion of EAPC in organic solvent, to
determine the optimum concentration for the esterification of
racemic ibuprofen using EAPC. EAPC was settled down
without AOT and with the addition of 10 mM AOT in the
middle of the reaction, whereas EAPC was well-dispersed at
higher concentrations of AOT (Figure 4). Furthermore, the
PANFs of EAPC when compared to EA and EAC can be
explained by the large amount of loaded LPs in PANFs. This
result matched well with the change in PANF thickness
observed by SEM and the LP loadings estimated from
elemental analysis. The specific activities of free LP, EA,
EAC, and EAPC were 12.0, 10.6, 6.1, and 6.8 mM min− mg
1
−1
of LPs, respectively. The lower specific activities of immobilized
enzymes can be explained by the combination of enzyme
inactivation during the enzyme immobilization and mass-
transfer limitation during the enzyme reaction.
The stability of EA, EAC, and EAPC was investigated
through shaking at 200 rpm at room temperature and 40 °C.
After 84 days of shaking at 200 rpm at room temperature,
EAPC retained 74% of the initial activity, whereas EA and EAC
retained 11 and 24% of initial activity, respectively (Figure 3).
The activity of free LP sharply decreased to less than 1% of
C
dx.doi.org/10.1021/la404189e | Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
was the result of high loading of LPs. Low amounts of LP in EA
and EAC were not effective on the esterification reaction of S-
(
+)-ibuprofen. Enantiomeric excess (ee) of EA, EAC, and
EAPC toward the S form of ibuprofen under 100 mM AOT
was calculated by the following equation:
1
4,26
ee (%) = ([S] − [R])/([S] + [R]) × 100
where [S] and [R] indicated the concentration of the S and R
forms of ibuprofen ester produced. The results obtained were
greater than 98% (see Figure S3 of the Supporting
Information), showing that EA, EAC, and EAPC can selectively
esterify S-(+)-ibuprofen to S-(+)-ibuprofen ester.
Recycled Use of EA, EAC, and EAPC for the
Esterification of (R,S)-Ibuprofen. EA, EAC, and EAPC
were recycled for repeated esterification of (R,S)-ibuprofen
(
Figure 6). After 9 repeated cycles, the results showed that
Figure 4. Dispersion and initial rates of EAPC dependent upon the
concentration of added AOT.
initial rates of esterification at various AOT concentrations were
investigated. The addition of 100 mM AOT resulted in the
highest activity, which was 61 times higher than that of the
control without AOT. This can be explained by the dispersion
of EAPC. AOT molecules present in the interface between the
hydrophilic enzyme surface and the hydrophobic isooctane aid
in better dispersion of EAPC, regardless of continuous water
production during esterification. Interestingly, the initial rate of
reaction decreased when the concentration of added AOT was
increased from 100 to 200 and 500 mM. This is likely due to
excessively high concentrations of AOT molecules, which can
Figure 6. Relative yield of S-(+)-ibuprofen ester with EA, EAC, and
EAPC at 40 °C with shaking (200 rpm).
25
inhibit the reaction between LP and substrate.
relative yields of EA, EAC, and EAPC were 5, 22, and 38%,
respectively. The decreased relative yield can be explained by
enzyme leaching while shaking (200 rpm) at 40 °C. The
relative yield of EA and EAC gradually decreased, while the
relative yield of EAPC stabilized after 6 cycles as a result of its
high stability.
Esterification of (R,S)-Ibuprofen. On the basis of the
AOT effect, we further investigated the esterification efficiency
of EA, EAC, and EAPC using 100 mM AOT, 10 mM
ibuprofen, 20 mM 1-propanol, and 1% (v/v) water. After 102 h
of esterification reaction, EA, EAC, and EAPC reached 1.2, 1.4,
and 42% conversion, respectively (Figure 5). For the initial rate
of esterification, EAPC had a 75-fold increased rate than EA
and EAC within 6 h. The rapid conversion observed in EAPC
CONCLUSION
■
The EAPC approach has demonstrated that large quantities of
LP can be immobilized and that LP can be highly stabilized for
a long-term under harsh conditions. Precipitation caused by
ammonium sulfate decreases the distance between LP
molecules and results in the effective cross-linking of LPs.
This is directly related to the long-term stability of immobilized
LPs in PANFs. EAPC also resulted in enhanced esterification of
ibuprofen in an organic solvent. Additionally, we induced better
dispersion of LPs in PANFs using AOT. The LP from C. rugosa
used in this work is a type of esterase, and there are many other
LPs that have useful catalytic reactions. Thus, this work
indicates that the EAPC approach could be applied to many
kinds of LPs in various fields based on successful LP
stabilization and application to ibuprofen resolution.
ASSOCIATED CONTENT
Supporting Information
LP loadings of EA, EAC, and EAPC (Table S1), volume ratio
and loading ratio of immobilized enzymes (Table S2), typical
GC chromatogram for the ibuprofen resolution (Figure S1),
■
*
S
Figure 5. Time course of ibuprofen conversion with EA, EAC, and
EAPC at 40 °C with shaking (200 rpm) for 102 h. The amount of
immobilization for each reaction was adjusted to use the same amount
of PANFs in each.
D
dx.doi.org/10.1021/la404189e | Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
stability of free LP, EA, EAC, and EAPC in aqueous buffer
under shaking (200 rpm) at 40 °C (Figure S2), and ee of EA,
EAC, and EAPC toward the S form of ibuprofen (Figure S3).
This material is available free of charge via the Internet at
http://pubs.acs.org.
(18) Foster, P. R.; Dunnill, P.; Lilly, M. D. Salting-out of enzymes
with ammonium sulphate. Biotechnol. Bioeng. 1971, 13, 713−718.
̌
̌
19) Mozhaev, V. V.; Melik-nubarov, N. S.; Sergeeva, M. V.; Siksnis,
(
V.; Martinek, K. Strategy for stabilizing enzymes. Part one: Increasing
stability of enzymes via their multi-point interaction with a support.
Biocatal. Biotransform. 1990, 3, 179−187.
(
20) Wu, S. H.; Guo, Z. W.; Sih, C. J. Enhancing the
AUTHOR INFORMATION
enantioselectivity of Candida lipase catalyzed ester hydrolysis via
■
noncovalent enzyme modification. J. Am. Chem. Soc. 1990, 112, 1990−
Corresponding Author
1
995.
*E-mail: jbkim3@korea.ac.kr.
(
21) Mustranta, A. Use of lipases in the resolution of racemic
ibuprofen. Appl. Microbiol. Biotechnol. 1992, 38, 61−66.
22) Goto, M.; Kamiya, N.; Miyata, M.; Nakashio, F. Enzymatic
Notes
(
The authors declare no competing financial interest.
esterification by surfactant-coated lipase in organic media. Biotechnol.
Prog. 1994, 10, 263−268.
ACKNOWLEDGMENTS
■
(23) Yu, H. W.; Wu, J. C.; Bun, C. C. Kinetic resolution of ibuprofen
catalyzed by Candida rugosa lipase in ionic liquids. Chirality 2005, 17,
This work was supported by the “International Collaborative
R&D Program” of the Korea Institute of Energy Technology
Evaluation and Planning (KETEP) grant funded by the Korea
government Ministry of Trade, Industry and Energy
20118510020020) and the National Research Foundation of
Korea (NRF) grant funded by the Korea government Ministry
of Science, ICT and Future Planning (2013K000229 and
1
(
6−21.
24) Monot, F.; Borzeix, F.; Bardin, M.; Vandecasteele, J. P.
Enzymatic esterification in organic mediaRole of water and organic
solvent in kinetics and yield of butyl butyrate synthesis. Appl. Microbiol.
Biotechnol. 1991, 35, 759−765.
(
(25) Gupte, A.; Nagarajan, R.; Kilara, A. Enzymatic oxidation of
cholesterol in reverse micelles. Ind. Eng. Chem. Res. 1995, 34, 2910−
922.
26) Moss, G. P. Basic terminology of stereochemistry. Pure Appl.
Chem. 1996, 68, 2193−2222.
“
2013, University−Institute Cooperation Program”).
2
(
REFERENCES
■
(
1) Azerad, R. Chemical biotechnologyBetter enzymes for green
chemistryEditorial overview. Curr. Opin. Biotechnol. 2001, 12, 533−
5
34.
(
2) Kim, J. B.; Grate, J. W.; Wang, P. Nanobiocatalysis and its
potential applications. Trends Biotechnol. 2008, 26, 639−646.
3) Koeller, K. M.; Wong, C. H. Enzymes for chemical synthesis.
Nature 2001, 409, 232−240.
4) Liese, A.; Villela, M. Production of fine chemicals using
biocatalysis. Curr. Opin. Biotechnol. 1999, 10, 595−603.
5) Zaks, A.; Klibanov, A. M. Enzyme-catalyzed processes in organic
solvents. Proc. Natl. Acad. Sci. U. S. A. 1985, 82, 3192−3196.
6) Kragl, U.; Eckstein, M.; Kaftzik, N. Enzyme catalysis in ionic
liquids. Curr. Opin. Biotechnol. 2002, 13, 565−571.
7) Gupta, M. N. Enzyme function in organic solvents. Eur. J.
Biochem. 1992, 203, 25−32.
8) Serdakowski, A. L.; Dordick, J. S. Enzyme activation for organic
solvents made easy. Trends Biotechnol. 2008, 26, 48−54.
9) Klibanov, A. M. Improving enzymes by using them in organic
solvents. Nature 2001, 409, 241−246.
10) Jaeger, K. E.; Eggert, T. Lipases for biotechnology. Curr. Opin.
Biotechnol. 2002, 13, 390−397.
11) Kim, J.; Grate, J. W.; Wang, P. Nanostructures for enzyme
stabilization. Chem. Eng. Sci. 2006, 61, 1017−1026.
12) Kim, B. C.; Zhao, X. Y.; Ahn, H. K.; Kim, J. H.; Lee, H. J.; Kim,
(
(
(
(
(
(
(
(
(
(
K. W.; Nair, S.; Hsiao, E.; Jia, H. F.; Oh, M. K.; Sang, B. I.; Kim, B. S.;
Kim, S. H.; Kwon, Y.; Ha, S.; Gu, M. B.; Wang, P.; Kim, J. Highly
stable enzyme precipitate coatings and their electrochemical
applications. Biosens. Bioelectron. 2011, 26, 1980−1986.
(
13) Fernandes, K. F.; Lima, C. S.; Lopes, F. M.; Collins, C. H.
Properties of horseradish peroxidase immobilised onto polyaniline.
Process Biochem. 2004, 39, 957−962.
(
14) Lee, G.; Kim, J.; Lee, J. H. Development of magnetically
separable polyaniline nanofibers for enzyme immobilization and
recovery. Enzyme Microb. Technol. 2008, 42, 466−472.
(
15) Kim, H.; Lee, I.; Kwon, Y.; Kim, B. C.; Ha, S.; Lee, J.-h.; Kim, J.
Immobilization of glucose oxidase into polyaniline nanofiber matrix for
biofuel cell applications. Biosens. Bioelectron. 2011, 26, 3908−3913.
(
16) Miller, C.; Austin, H.; Posorske, L.; Gonzlez, J. Characteristics of
an immobilized lipase for the commercial synthesis of esters. J. Am. Oil
Chem. Soc. 1988, 65, 927−931.
(
17) Huang, J. Syntheses and applications of conducting polymer
polyaniline nanofibers. Pure Appl. Chem. 2006, 78, 15−27.
E
dx.doi.org/10.1021/la404189e | Langmuir XXXX, XXX, XXX−XXX