Asymmetric electrosynthesis of amino acid using an electrode modified with amino acid oxidase and electron mediator

Article abstract of DOI:10.1246/cl.2000.110

Asymmetric synthesis of amino acid has been successfully achieved by electrochemical reduction of keto acid using an electrode on which amino acid oxidase and electron mediator are immobilized. The enantiomer excess closing to 100% was obtained.

Full text of DOI:10.1246/cl.2000.110

110
Chemistry Letters 2000
Asymmetric Electrosynthesis of Amino Acid Using an Electrode
Modified with Amino Acid Oxidase and Electron Mediator
Susumu Kawabata,* Naoko Iwata, and Hitoshi Yoneyama*
Department of Materials Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871
(Received October 8, 1999; CL-990864)
Asymmetric synthesis of amino acid has been successfully
achieved by electrochemical reduction of keto acid using an elec-
trode on which amino acid oxidase and electron mediator are
immobilized. The enantiomer excess closing to 100% was
obtained
Electrochemical utilization of enzymes has been extensively
studied from the viewpoint of fabrication of biosensors1-3 and
electroorganic synthesis systems.4,5 The most successful example
among them should be the glucose sensors prepared by using the
glucose oxidase (GOx), which have been already commercialized.
necessity to use the nicotinamide coenzyme (NAD(P)) and anoth-
To accomplish electron communication between the enzyme mol-
er enzyme catalyzing reduction of NAD(P) seems to be undesir-
ecules and an electrode, it is required to use electron mediators. In
able for preparation of an electrode equipped with all required
species.
the case of GOx having a flavin adenine dinucleotide (FAD) as a
redox active center, O2 is the original electron acceptor to induce
In order to induce the reverse reaction, it is essential to use
the enzyme reaction, i.e. oxidation of glucose, but some artificial
the electron mediator having a redox potential which is more neg-
redox species such as quinone derivatives and metal complexes
ative than that of FAD (–0.15 V vs. Ag/AgCl). It is also better for
also were found to make electron exchanges with FAD in the
enzyme.6 Such findings allowed preparation of reagentless glu-
cose sensors by immobilizing GOx together with those redox
species as electron mediators on an electrode.1,2
The amino acid oxidase (AOx) is another enzyme having
FAD as a redox active center. This enzyme catalyzes in-vivo oxi-
dation of amino acid to imino acid in the presence of O2.7 The
produced imino acid is hydrolyzed, giving keto acid and ammonia
in an aqueous solution, as given by the following reactions.
the electron mediator to possess an appropriate functional group
which is available for its chemical immobilization. With those in
mind we chose in this study 1-aminopropyl-1’-methyl-4,4’-dipyri-
dinium iodide (ADPy), which was prepared by quaternization of
4,4’-dipyridine with methyl iodide and 3-aminopropyl iodide. D-
Amino acid oxidase (D-AOx: Type X) from Procine Kedney and
L-amino acid oxidase (L-AOx: Type IV) from Croalus adaman-
teus venom were commercially available from SIGMA and used
as received. A glassy carbon plate (Tokai Carbon) having an
exposed area of 1.0 cm2 was used as an electrode substrate. Its
surface was successively polished with 1 and 0.3 µm alumina par-
ticles slurry to obtain the mirror-finished surface. The immobiliza-
tion of AOx and ADPy was conducted by using glutaraldehyde as
a cross-linking agent in a similar manner as the case of the immo-
bilization of other enzymes and electron mediators.2,3 Phosphate
buffer (pH 6.5, 0.1 ml) containing 10 nmol ADPy, 21.2 µmol glu-
taraldehyde, and 1 unit of D-AOx was cast on the electrode sur-
face, followed by drying for ca. 10 h in a glass vessel filled with
dry N2 at room temperature. The prepared electrode is denoted
here as D-AOx/ADPy/GC.
Hydrogen peroxide produced by the reaction (1) is decomposed
by the catalase in living organisms. If an appropriate electron
mediator is available, it is expected to induce electrochemical oxi-
dation of amino acid as the case of the glucose sensor.
Furthermore, since this enzyme reaction is known to proceed
under equilibrium conditions in the absence of the catalase,7,8 it is
also expected that the reverse reaction can be electrochemically
induced by choosing an electron mediator having the ability to
reduce FAD in AOx, as schematically shown in Figure 1. Based
on this idea, we have attempted in this study the electrochemical
reduction of keto acid to amino acid with the use of an electrode
on which both AOx and electron mediator are immobilized. In
this communication, it will be shown that the electrolysis using
the prepared electrode induces asymmetric production of amino
acid with the enantiomer excesses closing to 100%. The electro-
chemical conversion of keto acid to amino acid has already been
attempted by using the amino acid dehydrogenase.9 In that case,
Cyclic voltammetry of the D-AOx/ADPy/GC was taken in
0.1 mol dm-3 phosphate buffer (pH 6.5). The voltammogram
obtained at the potentials ranging between –0.7 V and 0 vs.
Ag/AgCl showed a couple of anodic and cathodic current peaks
that located at –0.23 and –0.43 V vs. Ag/AgCl, respectively. Since
the average of the peak potentials (–0.33 V vs. Ag/AgCl) was
well in accordance with the redox potential of ADPy (–0.34 V vs.
Ag/AgCl) dissolved in an aqueous solution, the redox waves
observed for the D-AOx/ADPy/GC electrode was assignable to
the redox reaction of ADPy immobilized on the electrode surface.
Integration of the reduction wave of the voltammogram allowed
estimation of the amount of ADPy on the electrode to be 9.8 nmol
Copyright © 2000 The Chemical Society of Japan

Chemistry Letters 2000
111
Figure 3, the reaction rate was enhanced by increasing the concen-
tration of substrates (i.e. pyruvic acid and NH4OH) and the
amount of AOx immobilized on the electrode. It is then suggested
that the reduction of substrates at AOx was the rate-determining
step under the condition chosen in this study.
which accorded well with that of ADPy which was mounted on
the electrode in the preparation of the electrode. The activity of D-
AOx immobilized on the electrode was determined by the chemi-
cal oxidation of D-alanine in the presence of O2 using the prepared
D-AOx/ADPy/GC as a catalyst. The obtained activity of 0.55 units
was smaller than the original activity of 1.0 unit, suggesting that
some inactivation of the enzyme molecules happened by their
immobilization.
Selective production of amino acid having L-configuration
was also successfully achieved by the electrochemical reduction
of phenylpyruvic acid using the L-AOx/ADPy/GC electrode. The
electrode was prepared by the same method as the case of the D-
AOx/ADPy/GC except for dissolution of 1 unit of L-AOx instead
of D-AOx in the casting solution. The electrolyte solution used
was 0.1 mol dm-3 phosphate buffer (20 ml) containing 30 mmol
dm-3 phenylpyruvic acid and 30 mmol dm-3 NH4OH. The electrol-
ysis at –0.7 V vs. Ag/AgCl for 10 h gave 8.5 mmol dm-3 L-pheny-
lalanine and no detectable D-phenylalanine.
The electrochemical production of L-alanine using L-
AOx/ADPy/GC and that of D-phenylalanine using D-
AOx/ADPy/GC were also attempted. However, significant
amount of products was not obtained. The L-AOx (Type IV) and
D-AOx (Type X) used in this study are known to possess the sub-
strate selectivity for phenylalanine and alanine, respectively, in the
original oxidation reaction. It is then likely that the substrate selec-
tivity of the enzymes also limits kind of the electrochemically
induced reverse reaction. If one would like to use this electrolysis
system to obtain several kinds of amino acids, enzymes having
lower substrate selectivity should be desirable. Experiments
focusing on such further exploration are underway.
The electrochemical reduction of pyruvic acid was conducted
with the use of the D-AOx/ADPy/GC electrode. The electrolysis
experiment was carried out using a two-compartment cell separat-
ed by a cation exchange membrane (Nafion 117, Aldrich). The
electrolyte solution used was 0.1 mol dm-3 phosphate buffer (pH
6.5, 20 ml) containing 30 mmol dm-3 pyruvic acid, 30 mmol dm-3
NH4OH. The identification of products and determination of their
amounts were made by using a high-performance liquid chro-
matography with a Sumichiral OA-5000 column. Figure 2 shows
the time course of alanine production obtained by the electrolysis
at –0.7 V vs. Ag/AgCl. As recognized, the product of D-alanine
increased with the electrolysis time, whereas no L-alanine was
produced, indicating that the electrochemical reduction of pyruvic
acid took place according to the reaction scheme shown in Figure
1. The HPLC analysis gave the enantiomer excess of 100% for
the production of D-alanine. The enantiomer excess larger than
99% was also obtained by the polarimetric analysis of the elec-
trolyte solution that was subjected to the electrolysis for 10 h. The
current efficiency larger than 97% was obtained through the elec-
trolysis experiment. Production of 8.9 mmol dm-3 D-alanine in 20
ml of the electrolyte solution allowed estimation of the turnover
number more than 36,000 for the immobilized ADPy. Those
results indicated clearly that the D-AOx immobilized on the elec-
trode kept its high selectivity for the asymmetric reaction and it
functioned well even for the reverse reaction induced by the elec-
tron mediation of the immobilized ADPy.
This research was supported by the Asahi Glass Foundation.
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The electrolysis experiments were conducted under some dif-
ferent conditions. Changes in the polarization potential between
–1.2 and –0.4 V vs. Ag/AgCl and changes in the amount of the
immobilized ADPy between 5 and 20 nmol did not influence sig-
nificantly the rate of D-alanine production. However, as shown in
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