ANALELE ŞTIINŢIFICE ALE UNIVERSITĂŢII “AL. I. CUZA” IAŞI
Tomul IV, s. Biofizică, Fizică medicală şi Fizica mediului 2008
LACCASE IMMOBILISED ON HYDROTALCITES
AS A 3rd GENERATION BIOSENSOR TYPE
Alina Manole1, D. Herea2, H. Chiriac2, V. Melnig1
KEYWORDS:
Laccase is one of the green enzymes that can directly reduce oxygen into water
under ambient conditions, while catalyze the transformation of a large number of
phenolic and non-phenolic aromatic compounds. The system laccase intercalated
in CoLDH clay with chitosan as enzyme linker seems to be appropriate as direct
electron transducer to Pt working electrode. The experimental conditions to
obtain current signal in terms of reproducibility and sensitivity have been
established as biosensor activity function of pH, temperature and hydroquinone
substrate concentration and specific working potential.
1.
INTRODUCTION
Laccases are cuproteins included in a small group of enzymes called blue
copper proteins or blue copper oxidases along with ascorbate oxidase and
ceruloplasmin. For the over 60 types of laccases isolated from different sources like
insects, plants, fungus and bacteria, differences in thermodynamic and kinetic
properties were observed, depending on the source, although the copper centre is
similar for all laccases. In general, laccases have, distributed among different binding
sites, 4 copper atoms classified in 3 types: types 1 and 2 are involved in electron
capture and transfer, while copper types 2 and 3 are involved in binding with oxygen.
The direct electron transfer in enzymes was first described for a laccase [1].
Laccases (E.C. 1.10.3.2, benzenediol:oxygen oxidoreductase) are phenol
oxidases that catalyses the oxidation of some inorganic and organic compounds
(especially phenols) with the concomitant electroreduction of oxygen to water:
O 2 4e   4H   2H 2 O
Thus, a molecular laccase layer attached at the surface of an electrode forms an
oxygen transducer [2]. The electron transfer and reduction of 2 oxygen atoms to water
mechanism are not yet fully understand in the case of laccases.
In a typical reaction catalysed by laccase, a phenolic substrate is subjected to a
one-electron oxidation giving rise to an aryloxyradical. This active species can be
converted to a quinone in the second stage of the oxidation. The quinine, as well as the
free radical product undergoes non-enzymatic coupling reactions leading to
1
2
“Al.I. Cuza” University, Faculty of Physics, Carol I Blvd., No.11, 700506, Iasi
National Institute of Research and Development for Technical Physics 47 Mangeron Blvd., 700050, Iasi
12
Alina Manole, D. Herea, H. Chiriac, V. Melnig
polymerization. Simple diphenols such as hydroquinone and catechols are good
substrate for the majority of laccases [3].
Fungal laccases, in free form or immobilized, but also in organic solvents, were
used in different biotechnological and environment applications like biobleaching,
waste water treatment, biosensors, biofuel cells as cathodic biocatalyser,
delignification and demethylation. Laccase biosensors are an alternative to phenolic
compounds determination besides using peroxidases, for which the background current
produced by H2O2 is limiting the biosensor sensitivity [4].
2.
MATERIALS AND METHODS
Laccase (EC 1.10.3.2, benzenediol:oxygen oxidoreductase, from trametes
versicolor), hydroquinone and chitosan were purchased from Sigma Aldrich. They
were used as received, without further purification.
The hydrotalcites (CoLDHs) were prepared as follows: an aqueous solution (85
ml) of Mg(NO3)2·6H2O ((0.03-q) mol)/Al(NO3)3·9H2O (0.01mol)/ Co(NO3)2·3H2O (q
mol, 0.005  q  0.01) and an aqueous solution of Na2CO3 (1M, 30 ml) were added
dropwise together over a period of 2 h at a constant pH value of 8.2. The resulting
precipitate was aged at 305 K for 24 h under stirring. The samples were calcined under
air at 723 K; the temperature was raised with 8 K·min-1 to 723 K, maintained there for
5 hours and then cooled slowly under nitrogen.
All other chemicals are of analytical grade. 0.1M phosphate buffer (PBS)
consisted of Na2HPO4 and NaH2PO4 and 0.1M Britton buffer solutions, obtained by
mixing the same amount of 0.1M acetic acid, boric acid and phosphoric acid were
employed as supporting electrolyte. The desired Britton buffer solutions pH was adjusted
by different amounts of NaOH solution. All solutions were made using deionised water.
Pt electrode was washed ultrasonically in water and ethanol for a few minutes,
respectively. A 1% chitosan solution was prepared by dissolving chitosan in 2% acetic
acid in Britton buffer with pH 5 with magnetic stirring for about 2 h. For preparation of
laccase/CoLDHs –chitosan/Pt electrode, 3mL of 1% chitosan solution was mixed with
4mg laccase and 8 mg CoLDHs. The laccase is firstly dissolved in 0.5 ml deionized
water. Then 10 μL of the laccase/ CoLDHs –chitosan mixture was spread onto the Pt
electrode surface with a pipette. Finally, the modified electrode was allowed to dry
over night at 50◦C.
Chitosan was used for enzyme immobilization because it is an inert,
biocompatible and biodegradable amphiphilic polymer [5] and a special feature of
amphiphilic polymers is the formation of association structures in aqueous solution.
The unique structural feature of chitosan is the high content of primary amines, and
these amines confer important functional properties to chitosan that can be exploited.
At low pH, chitosan is a water-soluble cationic polyelectrolyte due to these protonated
amines. At high pH, chitosan’s amines become deprotonated and the polymer loses its
charge and becomes insoluble. In this case, chitosan’s electrostatic repulsions are
reduced allowing the formation of inter-polymer associations (liquid crystalline
domains or network junctions). As a result, fibers, films, or hydrogels can be obtained,
depending on the conditions used to initiate the soluble-insoluble transition. The
soluble-insoluble transition occurs at pH between 6 and 6.5, which is a particularly
LACCASE IMMOBILISED ON HYDROTALCITES…
13
convenient range for biological applications [6]. In this case the entrapping of enzyme
into the composite film is done without using a cross-linking reagent.
The FT-IR spectra were recorded with a 6100 JASCO spectrometer with 4 cm-1
resolution in the range 4000-400 cm-1.
Cyclic voltammetry (CV) and chronoamperometry measurements were carried
out on a VoltaLab 10 (PGZ 100) potentiometer controled by VoltaMaster 4 software.
Hydroquinone solutions in phosphate buffer pH 7 were used as electrochemical probes.
A conventional three-electrode setup was used. The modified electrode was applied as
the working electrode. Two identical 3 mm diameter platinum electrodes were used as
working (WE) and counter (CE) electrodes and Ag/AgCl electrode as reference
electrode (REF), respectively. All the voltammograms were performed at 20mV/s scan
rate. The chronoamperometry measurements were performed at constant potential (360
mV). Before each electrochemical measurement, the Pt electrodes system was
immersed in HNO3 50% v/v solution for 1 min to remove the deposits on the surface
electrodes and then it was washed with distillated water and dried thus fresh and clean
surfaces could be obtained. All electrochemical measurements were performed at room
temperature. For testing the answer of the modified electrode at pH variation the
hydroquinone solutions were prepared in Britton buffer.
3.
RESULTS AND DISSCUTIONS
3.1 FT-IR spectra
The FT-IR spectra show noticeable differences both in the functional group
region, and fingerprint region. Inspection of the functional group region and
correlation chart reveals characteristic bands of N – H and C – N groups of peptide
bond, C = O and N – H bands from amide and O – H from hydroxyl.
895
CH
Transmittance (%)
80
2878
CH
60
40
80
2930
Laccase
2885
M
2928
M
3438
LDH
20
3400
3200
Wavenumber (cm-1)
3000
2800
1263
CH
40
0
3600
1262
CH
1156
CH
895
CH
1639
CH
1649
Laccase
1642
CH+Laccase
1800
1600
792
1088
LDH
CH
786
804
Laccase
Laccase LDH
1156
CH
1089
CH
1400
1200
LDH
555
LDH
667
809
Laccase LDH
60
20
0
3800
Chitosan (CH)
Laccase
LDH
Chitosan, Laccase, LDH (M)
100
Transmittance (%)
100
Chitosan (CH)
Laccase
LDH
Chitosan, Laccase, LDH (M)
657
LDH
M
1000
560
LDH
800
600
450
LDH
454
LDH
400
Wavenumber (cm-1)
(a)
(b)
Fig. 1 FTIR spectra a) functional group region and b) fingerprint region, for 1%
chitosan solution prepared in 2% acetic acid in britton buffer pH 5, laccase, LDH and
1% chitosan solution mixed with 4mg laccase and 8 mg LDH.
14
Alina Manole, D. Herea, H. Chiriac, V. Melnig
Strong water absorption bands are within 3900 – 2800 cm-1 and 1750 – 1550
cm-1, representing the stretching and deformation vibration of O – H group in water,
respectively (the fundamental stretching vibrations mode of water occur within the
3900–2800 cm-1 region and the inplane bending mode at around 1640 cm-1.
Even tough in the fingerprint region the spectrum is more complex, the general
idea is that the result is a physical mixture of laccase, LDH and chitosan in solution.
3.2 Electrochemistry of redox-active system
A redox-active system can be characterized by electrochemical measurements. A
typical cyclic voltammogram is shown in Figure 2a for the case of an unmodified working
electrode immersed in a non-stirred solution containing different hydroquinone
concentrations as the electroactive species in phosphate buffer pH 7. The potential axis is
versus an Ag/AgCl REF electrode. The switching potential has been set at-300 mV to
+500 mV vs. REF etectrode and was selected to be positioned on either side of the main
redox peaks. The reduction shows a cathodic peak at +360 mV and an anodic peak at -70
mV resulting from re-oxidation of the reduction product at reverse potential scan, booth
dependent of the hydroquinone concentration.
(a)
(b)
Fig. 2 Cyclic voltammograms for hydroquinone solutions with different concentrations
using unmodified Pt electrode (a) and cyclic voltammograms for 0.8 mM
hydroquinone solutions using unmodified and chitosan modified Pt electrode,
respectively (b).
The progress of assembling system can be monitored by addition of mixture
components on the WE electrode. In Fig. 2b the influence of the chitosan on the double
layer of the WE are shown; redox process is increased while the oxidation is not.
Differences in the redox processes seen do not appear at normal pH by the
presence of laccase and anionic clay in the system (Fig. 3a and 3b). The
chronoamperometry measurements demonstrate that in the presence of anionic clay the
current densities increase (Fig. 4).
Major changes are recorded in redox peaks positions (Fig. 5) and current
densities response (Fig. 6) when the experiments are performed as functions of pH and
LACCASE IMMOBILISED ON HYDROTALCITES…
15
temperature bath solutions. The optimum working parameter conditions for laccase
immobilized CoLDH for hydroquinone biosensor seem to be 70˚ C degree and pH 4.
(a)
(b)
Fig. 3 Cyclic voltammograms for hydroquinone solutions with different concentrations
using chitosan and laccase modified Pt electrode (a) and cyclic voltammograms for
hydroquinone solutions with different concentrations using chitosan, laccase and
anionic clay modified Pt electrode.
pH=4
pH=5
pH=6
pH=7
0,5
Current density (mA/cm2)
Current density (mA/cm2)
Fig. 4 The current answer of the chitosan and laccase and chitosan, laccase and anionic
clay modified Pt electrode, respectively, with hydroquinone concentration variation.
0,0
0,0
-0,5
-0,5
-400
pH=4
pH=5
pH=6
pH=7
0,5
-200
0
200
400
Potential (mV)
(a)
600
800
-400
-200
0
200
400
Potential (mV)
(b)
600
800
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Alina Manole, D. Herea, H. Chiriac, V. Melnig
Fig. 5 Cyclic voltammograms for 0.8 mM hydroquinone solutions at different pH in
Britton Buffer solutions using chitosan and laccase modified Pt electrode (a) and
chitosan, laccase and anionic clay modified Pt electrode.
0,14
Current density (mA/cm2)
0,12
0,10
0,08
0,06
0,04
0,30
Pt electrode modified
with chitosan and laccase
Pt electrode modified
with chitosan, laccase
and anionic clay
0,28
Current density (mA/cm2)
Pt electrode modified with
chitosan and laccase
Pt electrode modified with
chitosan, laccase and anionic clay
0,26
0,24
0,22
0,20
0,18
0,16
0,14
0,12
0,02
4
5
6
7
0,10
20
30
pH
40
50
60
70
80
90
Temperature (0C)
(a)
(b)
Fig. 6 Answer of the chitosan and laccase and chitosan, laccase and anionic clay
modified Pt electrode, respectively, with pH variation (a) and with temperature
variation (b).
4.
CONCLUSIONS
Free and immobilized laccase/CoLDHs–chitosan/Pt electrode was studied for
biosensor activity function of pH, temperature and hydroquinone substrate
concentration and specific working potential. The system laccase intercalated in
CoLDH clay with chitosan as enzyme linker increase the response in current of the
biosensor. The optimal working conditions for this being 70˚ C degree and pH 4.
Acknowledgement
This research was supported by CEEX 1639/2006 (BIOSNANOMAG)
research program.
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