Enhanced enzymatic reaction of tyrosinase-immobilized polyacrylamide- (γ-cyclodextrin) membrane coated on a platinum disk electrode in acetonitrile

Article abstract of DOI:10.1246/cl.2007.506

A functionalized stable film, polyacrylamide-(γ-cyclodextrin) (PAA-γ-CD), synthesized from polyacrylamide (PAA) and γ-cyclodextrin (γ-CD), was used to immobilize enzyme. The membrane containing γ-CD provided an excellent environment for enzymatic kinetics

Full text of DOI:10.1246/cl.2007.506

506
Chemistry Letters Vol.36, No.4 (2007)
Enhanced Enzymatic Reaction of Tyrosinase-immobilized Polyacrylamide–(ꢀ-Cyclodextrin)
Membrane Coated on a Platinum Disk Electrode in Acetonitrile
Toshio Nakamura,ꢀ Xueping Ji, Kazuyoshi Endo, and Daisuke Takano
Department of Chemistry, Faculty of Science, Shinshu University, Matsumoto 390-8621
(Received December 20, 2006; CL-061487; E-mail: toshion@shinshu-u.ac.jp)
A functionalized stable film, polyacrylamide–(ꢀ-cyclodex-
trin) (PAA–ꢀ-CD), synthesized from polyacrylamide (PAA)
and ꢀ-cyclodextrin (ꢀ-CD), was used to immobilize enzyme.
The membrane containing ꢀ-CD provided an excellent environ-
ment for enzymatic kinetics between tyrosinase in the membrane
and the substrates in acetonitrile.
AN solution. In the absence of the substrates, no detectable sig-
nal was observed for any of the electrodes. Nor was any response
observed at the Tyro-free electrodes. On addition of substrates,
however, reduction peaks were observed at the Tyro-containing
electrode, which resulted from the reduction of o-quinone
produced by the enzymatic reaction.7,8
OH
O
O
OH
Tyrosinase
Previous studies have confirmed that enzymes remain active
in organic solvents containing little or no water.1–3 The use of
enzymes in nonaqueous solvents has a number of advantages,
such as high solubility of hydrophobic substrates, enhanced
thermostability, the suppression of various side reactions, altered
enzymatic selectivity, and the comparative simplicity of immo-
bilization procedures. PAA has been used previously for enzyme
immobilization.4 Hydrophilic non-cross-linked and non-plasti-
cized PAA has been used in our laboratory as a matrix for bio-
functional material immobilization.5 It has been confirmed that
the PAA-membrane-modified electrode yields microelectrode
ensembles.6 The kinetic properties of a tyrosinase-immobilized
PAA electrode in acetonitrile (AN)7 and in N,N-dimethylacet-
amide8 have been reported. These studies have shown that the
PAA membrane offers a matrix for biofunctional material immo-
bilization. However, the sensitivity of the reaction between
enzymes and substrates are expected to improve further with
respect to response time and sensitivity. Since cyclodextrin
(CD) possesses a hydrophobic internal cavity and a hydrophilic
exterior, it seems promising as an electrode modifier for enzy-
matic kinetics. It has been widely studied due to its ability
to form inclusion complexes with a large variety of organic
molecules.9 Its well-known ability to form supramolecular
complexes with suitable substances has resulted in its use to
enhance electrode selectivity.10 It appears likely that the
combination of PAA and ꢀ-CD might provide an excellent
means of improving the function of enzyme electrode. In the
present study, a stable film, PAA–ꢀ-CD was used in the immo-
bilization of enzyme, tyrosinase (Tyro), on a platinum electrode.
It was expected that the electrode modification would construct
enzymatic reaction sites on the PAA membrane between the sub-
strates, catechol and 4-methylcatechol in dipolar aprotic solvents
and Tyro in the membrane. PAA–ꢀ-CD was synthesized by an
esterification of carboxyl-modified PAA and ꢀ-CD using lipase
as a catalyst in n-heptane at 50 ꢁC during three days as that in
similar studies.11 The mass ratio between PAA and ꢀ-CD was
7.5:1 at the present study.
+ H2O
+ 1/2O2
1,2-benzoquinone
catechol
As can be seen in Figure 1, the peak currents were much
larger at Pt/(PAA–ꢀ-CD + Tyro) than at Pt/(PAA + Tyro),
which shows that ꢀ-CD played an important role in enhancing
enzymatic activity. The typical cyclic voltammograms for 4-
methylcatechol are shown in Figure 1. The voltammogram at
the Pt/(PAA–ꢀ-CD + Tyro) electrode showed a more positive
reduction peak potential (Epc) than that obtained at the Pt/
(PAA + Tyro) electrode, as can be seen in Table 1.
Figure 2 shows the response behavior of the ꢀ-CD-modified
electrodes obtained by an amperometric method. On adding sub-
strates, no response was observed for the Tyro; Pt/PAA–ꢀ-CD
(Figure 2a) and Pt/PAA (Figure 2b), however the enzyme-con-
taining electrode responded rapidly and reached a steady-state
current. The response time and sensitivity for the Pt/(PAA–ꢀ-
CD + Tyro) electrode (Figure 2d) improved remarkably com-
pared with the ꢀ-CD-free enzyme electrodes. These results must
be due to the effect of ꢀ-CD; hydrophobic internal cavity and
hydrophilic exterior, at the PAA membrane on the enzymatic
reaction between Tyro and substrates.
Figure 3 shows the calibration plot of the Pt/(PAA–ꢀ-
A
0.2 µA
B
-1.0
-0.5
0.0
E / V vs. Ag/Ag+
The reactivity of the Pt/(PAA–ꢀ-CD + Tyro) electrode
with catechol and 4-methylcatechol was compared with similar
systems without ꢀ-CD.
The voltammetric responses of the Pt/(PAA–ꢀ-CD +
Tyro), Pt/PAA–ꢀ-CD, Pt/(PAA + Tyro), and Pt/PAA elec-
trodes were measured for two substrates mentioned above in
Figure 1. Cyclic voltammograms of 25 mM 4-methylcatechol
in 50 mM Et4NClO4–AN solution at (A) Pt/(PAA–ꢀ-CD +
Tyro), and (B) Pt/(PAA + Tyro) electrodes. Dotted and solid
lines are for blank and sample solutions, respectively. Scan rate:
0.10 V/s.
Copyright ꢀ 2007 The Chemical Society of Japan

Chemistry Letters Vol.36, No.4 (2007)
507
Table 1. Electrochemical parameters of the tyrosinase elec-
trodes (Pt/(PAA–ꢀ-CD + Tyro) (E1) and Pt/(PAA + Tyro)
(E2)) in 50 mM Et4NClO4 AN solution at 35 ꢁC
d
30 nA
Epc/V vs
Fc/Fcþ
Km/mM Imax/mA cmꢂ2
6 min
Substrate
7
7
E1
ꢂ0:60 ꢂ0:70 5.6 6.6
4-Methylcatechol ꢂ0:62 ꢂ0:75 6.1 5.9
E2
E1 E2
E1
E2
c
Catechol
7.5
15.3
4.4
9.3
b
for the enzyme electrodes are somewhat variable. However, no
remarkable differences in Km, were observed for these substrates
either at the ꢀ-CD containing or the ꢀ-CD-free electrode.
In conclusion, the Tyro-immobilized PAA–ꢀ-CD provided
to be an excellent enzyme electrode. This report has described
how the ꢀ-CD containing PAA membrane provided preferable
better environment for enzyme kinetics. The PAA–ꢀ-CD with
Tyro attached promoted higher enzymatic activity on reaction
with catechol and 4-methylcatechol in AN. The results must
be due to the CD’s effect on the circumstance at the PAA
Tyro-immobilized membrane.
a
Time
Figure 2. Typical current–time response of (a) Pt/PAA–ꢀ-CD,
(b) Pt/PAA, (c) Pt/(PAA + Tyro), and (d) Pt/(PAA–ꢀ-
CD + Tyro) electrodes on the stepwise additions of catechol
from 0.90 to 13 mM in 50 mM Et4NClO4–AN solution at
ꢂ0:70 V vs Ag/Agþ.
5
It is very interesting to know that how to work the ꢀ-CD to
the enzymatic reaction and/or to enhancement of the electrode
response. Futher investigation both from the view point of
molecular dynamics using nmr and from other CDs are now in
progress in our research group.
4
0.4
3
0.3
0.2
2
0.1
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0.0
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Figure 3. Calibration plot of the Pt/(PAA–ꢀ-CD + Tyro)
electrode for catechol in 50 mM Et4NClO4–AN solution at
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Published on the web (Advance View) March 3, 2007; doi:10.1246/cl.2007.506