8
8
K. Min et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 87–90
where F is Faraday constant, S is surface area of electrode, C is
concentration of l-DOPA, D is diffusion coefficient, v is scan rate
in cyclic voltammogram, R is ideal gas constant, T is temperature,
n = 2 meant charge transfer number, and ϕ = 0.1 is calculated by
Nicolson method [10,11].
ꢀ
ꢁ
1/2
Fv
RT
1/2
ip = 0.496 ∗ F ∗ S ∗ C ∗ D
∗
(1)
(2)
ꢀ
ꢁ
1/2
nꢀFD
RT
ke =
ϕ v1/2
2.3. Current response
The current response was investigated using 3-electrode sys-
Scheme 1. Schematic diagram of electroenzymatic l-DOPA synthesis (anode: ELAT
with 20% Pt wire, cathode: tyrosinase/carbon nanopowder/polypyrrole composite
electrode).
tem. Glassy carbon, ELAT, and Ag/AgCl were used as the working,
counter, and reference electrode, respectively. 1 mM of l-DOPA was
used as the substrate. When the current in the system was sta-
bilized, tyrosinase was added and then the current response was
investigated.
Herein, we used well-dispersed l-tyrosine in order to increase
the substrate concentration in aqueous phase and then achieved
the improving productivity. Also we reported the reason for the
high conversion rate achieved in the electro-enzymatic system by
determining the kinetic parameters.
3. Results and discussion
3.1. Electroenzymatic l-DOPA synthesis using well-dispersed
l-tyrosine as a substrate
2
. Materials and methods
The electro-enzymatic system achieved the excellent conver-
sion rate in l-DOPA synthesis [9], but the productivity in the system
is still limited due to the low solubility of the substrate, l-tyrosine,
Tyrosinase (E.C. 1.14.18.1) from mushroom purchased from
Sigma–Aldrich. Unless otherwise stated, all chemicals were pur-
chased from Sigma–Aldrich at the highest grade available and were
used without further purification. ELAT with 20% Pt and Ag/AgCl
electrode were purchased from WonATech (Korea). All electro-
chemical experiments were controlled by Autolab PGSTAT 302 N
potentiostat/galvanostat (Ecochimie, Netherland).
<
2.5 mM. In order to increase the solubility, various water-miscible
organic solvents were tested, but the solubility of l-tyrosine was not
significantly changed (data not shown). So, as a solution for the low
solubility of L-tyrosine, we perceived that emulsification can some-
times be an alternative for the insoluble or poor soluble chemical
in the reaction phase [12,13]. For example, phytosterols, which can
significantly reduce cholesterol levels in human beings, is insolu-
ble in water and poorly soluble in oil. To increase the amount of the
phytosterol in functional foods, an emulsification was formed by
esterifying phytosterol [13,14]. Herein, we prepared well-dispersed
l-tyrosine solution by using the wet-milling method to aim for
overcoming the solubility limitation of l-tyrosine and then improv-
ing the productivity in the electroenzymatic l-DOPA synthesis.
The overall results of l-DOPA productions for various concen-
trations of well-dispersed l-tyrosine are summarized in Table 1.
Initially, the electroenzymatic l-DOPA synthesis was carried out
using 1 mM solution of well-dispersed l-tyrosine. As a result, the
substrate was almost fully (99.8%) converted to l-DOPA within
30 min at the composite electrode of size (1 cm × 1 cm × 0.6 cm).
This implies that the well-dispersed l-tyrosine sample, which can
supply the concentrated substrate in aqueous phase, is converted
2
.1. l-DOPA synthesis using well-dispersed l-tyrosine
Well-dispersed l-tyrosine was prepared by the wet-milling
method referred to in the US patent 5145684. Zirconium oxide and
polyvinylpyrrolidinone K-30 were used as milling media and sur-
factant, respectively. The reaction mixture including the milling
media, the surfactant, and l-tyrosine was incubated for 72 h at
ambient temperature, while shaking at 20 rpm. After the reaction
had reached completion, the milling media was removed by filtra-
tion. The concentration of the prepared well-dispersed l-tyrosine
solution was determined after filtration using Bradford reagent.
The 3-dimensional tyrosinase/carbon nanopowder/polypyrrole
composite electrode was prepared in accordance with the pre-
vious report [9]. All L-DOPA synthesis were conducted in batch
reactor with 60 mL of well-dispersed l-tyrosine in phosphate
buffer (pH 6.5). The 3-dimensional tyrosinase/carbon nanopow-
der/polypyrrole composite electrode, ELAT with 20% Pt, and
Ag/AgCl were used as the working, counter, and reference elec-
trode, respectively. The cathodic potential was maintained at
−
1
−1
quickly to l-DOPA with a productivity of 0.39 g L
h and thus
our electroenzymatic system was expected to be used at various
concentrations of well dispersed l-tyrosine by suitably increas-
ing the electrode size and enzyme concentration. So, we gradually
increased concentration of the well-dispersed l-tyrosine up to
500 mM by suitably increasing electrode size. As shown in Table 1,
the process resulted in 77.7% conversion rate and the productiv-
−
530 mV vs. Ag/AgCl during the reaction. The synthesized l-DOPA
was analyzed by modified Arnow method as previously reported
9].
[
−
1
−1
ity also dramatically increased to 15.3 g L
h due to the rapid
2
.2. Kinetic study
reduction of DOPAquine to l-DOPA again by the steady electron
supply from the working cathode as discussed in Section 3.2. Also,
to the best of our knowledge, there has been no report on l-DOPA
production with such outstanding productivity (Table 2). For all
the different concentrations of well-dispersed l-tyrosine, there
was no brown precipitate, formed by spontaneous oxidation of
DOPAquinone, indicating that the modified electrode was efficient
in electron transfer.
To determine the reaction constants of tyrosinase catalysis
by Lineweaver–Burk plot, l-tyrosine (0.1–1.0 mM) and of l-DOPA
0.1–1.0 mM) in phosphate buffer (pH 6.5) were used as a substrate
(
for cresolase and catecholase activity, respectively.
The reaction constant for the electrical reduction of
DOPAquinone to l-DOPA was calculated by following equations,