Highly efficient and regioselective production of an erythorbic acid glucoside using cyclodextrin glucanotransferase from Thermoanaerobacter sp. and amyloglucosidase

Article abstract of DOI:10.1016/j.molcatb.2013.03.011

In order to continuously supply erythorbic acid (EA) for long-term cell cultures, we synthesized a stable EA derivative, 2-O-α-d-glucopyranosyl-d- erythorbic acid (EA-2G), as a useful tool for analyzing the biological function of EA. The specific and effi

Full text of DOI:10.1016/j.molcatb.2013.03.011

Journal of Molecular Catalysis B: Enzymatic 92 (2013) 19–23
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Highly efficient and regioselective production of an erythorbic acid glucoside
using cyclodextrin glucanotransferase from Thermoanaerobacter sp. and
amyloglucosidase
Akihiro Taia,∗, Yuji Iwaokaa, Hideyuki Itob
a Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023, Japan
b Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to continuously supply erythorbic acid (EA) for long-term cell cultures, we synthesized a sta-
ble EA derivative, 2-O-␣-d-glucopyranosyl-d-erythorbic acid (EA-2G), as a useful tool for analyzing the
biological function of EA. The specific and efficient production process of EA-2G consisted of two steps:
transglycosylation by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. and hydrol-
ysis by amyloglucosidase from Aspergillus niger. EA-2G was regioselectively formed by CGTase using EA
and ␥-cyclodextrin in pH 4.0 acetate buffer at 40 ◦C for 24 h. It seemed that several EA-2-oligoglucosides
were also formed in this reaction mixture. Additional hydrolysis at 60 ◦C for 2 h of the reaction mixture
by glucoamylase resulted in efficient production of EA-2G. EA-2G was obtained in two steps in 49.1%
overall yield from EA.
Received 11 December 2012
Received in revised form 21 February 2013
Accepted 20 March 2013
Available online xxx
Keywords:
Erythorbic acid
Transglucosylation
Cyclodextrin glucanotransferase
Amyloglucosidase
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
the instability of AA does not permit continuous supply to in vitro
cell culture with AA. AA-2G markedly enhanced sheep-red-blood-
l-Ascorbic acid (AA, Fig. 1), known as vitamin C, plays key
roles in many biological processes, such as collagen formation,
carnitine synthesis, and iron absorption [1,2]. In addition, it is an
important antioxidant in food and biological systems [3], but AA
is unstable under various oxidative conditions such as exposure
to neutral pH, heat and heavy metals, resulting in rapid degrada-
tion [4]. Yamamoto et al. have developed a stable AA derivative,
2-O-␣-d-glucopyranosyl-l-ascorbic acid (AA-2G, Fig. 1), from AA
and maltose or other ␣-glucans by a regioselective transgluco-
sylation with ␣-glucosidases from digestive organs of mammals
and rice seed and with cyclodextrin glucanotransferase (CGTase)
from Bacillus stearothermophilus [5–8]. AA-2G exhibits vitamin C
activity in vitro and in vivo after enzymatic hydrolysis to AA by
␣-glucosidase [9–11]. AA-2G has been approved by the Japanese
Government as a quasi-drug principal ingredient in skin care and
as a food additive and is now widely used as a medical additive in
commercial cosmetics.
cell-specific plaque-forming cell responses in cultured murine
splenocytes [15,16] and mitogen-induced IgM and IgG production
in human peripheral blood lymphocytes [17]. A single addition of
AA caused no increase in the antibody responses, but repeated addi-
tions of AA throughout the culture period enhanced the responses
to the same level as that induced by a single addition of AA-2G.
The enhancing effect of AA-2G was abrogated in the presence of
castanospermine, an ␣-glucosidase inhibitor. These results sug-
gest that AA-2G is capable of enhancing antibody production in
cultured splenocytes via continuous supplementation of AA. There-
fore, AA-2G can be used for long-term cell cultures as a useful tool
for analyzing the biological function of AA.
d-Erythorbic acid (EA, Fig. 1) (synonyms: d-isoascorbic acid, d-
araboascorbic acid) is a synthetic C-5 epimer of AA [18]. It has been
reported that EA has chemical properties very similar to those of
AA and is widely used as a food antioxidant in processed foods
[19] and that EA shows only one-twentieth of the antiscorbutic
activity of AA in guinea pigs [20]. Recently, it has also been reported
that the enhancing effect of EA on iron absorption from ferrous
sulfate exceeds that of AA in humans [21]. However, little is known
about the physiological function of EA. EA seems not to be useful
for long-term cell cultures for analyzing the biological function of
EA, since EA is unstable under various oxidative conditions as is AA.
We assumed that EA could be continuously supplied to an in vitro
cell culture by using a stable EA derivative in a similar manner as
AA has been reported to enhance immune responses such as
lymphocyte proliferation [12], neutrophil function [13] and chemo-
taxis of leukocytes [14]. However, there are few data on the effect
of AA on in vitro antibody production. This seems to be because
∗
Corresponding author. Fax: +81 824 74 1779.
E-mail address: atai@pu-hiroshima.ac.jp (A. Tai).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.03.011

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A. Tai et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 19–23
into a 2.0 ml screw cap micro tube (Fukae Kasei, Kobe, Japan), after
which 950 ␮l of 50 mM acetate buffer (pH 4.0–5.5) was added. After
addition of Toruzyme 3.0 L (50 ␮l), the mixture was placed in a
thermoregulated shaker (M·BR-022, TAITEC, Saitama, Japan) and
was stirred at 1100 r/min for 24 h at 35–60 ◦C. After 24 h, the reac-
tion mixture was diluted 100-fold with 80% acetonitrile–50 mM
ammonium acetate and then centrifuged. Ten ␮l of the resulting
supernatant was subjected to HPLC analysis. HPLC analysis was
carried out by a modification of our previous method [23]. Separa-
tion for transglycosylation products of EA was achieved by isocratic
elution on an Inertsil Diol column (ꢀ 4.6 mm × 250 mm, 5 ␮m, GL
Sciences Inc., Tokyo) kept at 40 ◦C with 80% acetonitrile–50 mM
ammonium acetate at a flow rate of 0.7 ml/min. The absorbance
at 260 nm was monitored. The reaction yield of monoglucosy-
lated products of EA was determined quantitatively as AA-2G
equivalent.
2.4. Addition of amyloglucosidase to the glycosylation reaction
mixture
Fig. 1. Chemical structures of AA, AA-2G, EA and EA-2G.
described above by using AA-2G and that the stable EA derivative
could be used as a useful tool for analyzing the biological function
of EA. It has been reported that a stable EA derivative, 2-O-␣-d-
glucopyranosyl-d-erythorbic acid (EA-2G, Fig. 1), was synthesized
from EA and ␣-cyclodextrin by a transglucosylation with CGTase
from B. stearothermophilus [22]. This synthesis was fundamentally
based on the method of AA-2G synthesis [7], and the yield (ca. 10%)
of EA-2G was lower than that (ca. 40%) of AA-2G. Hence, in this
study, we established conditions for specific and efficient formation
of EA-2G by using commercially available enzymes, and we found
that this method is superior to the previous method in terms of
reaction efficiency in large-scale production.
EANa monohydrate (30 mg, 139 ␮mol) and ␣- or ␥-cyclodextrin
(60.5 ␮mol) were put into a 2.0 ml screw cap micro tube, after
which 950 ␮l of 50 mM acetate buffer (pH 4.0) was added. After
addition of Toruzyme 3.0 L (50 ␮l), the mixture was placed in a
thermoregulated shaker and was stirred at 1100 r/min for 24 h at
40 ◦C. After 24 h reaction, the temperature of the reaction mixture
was raised to 60 ◦C and 50 ␮l of amyloglucosidase (1000 units/ml)
was added. Then the resulting mixture was stirred at 1100 r/min
for 8 h at 60 ◦C. Aliquots of 50 ␮l were withdrawn at the indicated
times, diluted 100-fold with 80% acetonitrile–50 mM ammonium
acetate, and then centrifuged. Ten ␮l of the resulting supernatant
was subjected to HPLC analysis.
2. Experimental
2.5. Preparation and purification of
2-O-˛-d-glucopyranosyl-d-erythorbic acid
2.1. General experimental procedure
1H NMR, 13C NMR and 2D NMR spectra were recorded on a
Varian NMR System 600 MHz instrument. Electron spray ioniza-
tion (ESI) high-resolution mass spectra were obtained on a Bruker
Daltonics MicrOTOF II instrument using direct sample injection.
Optical rotations were obtained at JASCO DIP-1000. The HPLC anal-
yses were carried out with a system consisting of a Hitachi L-2130
pump, L-2420 UV-VIS detector, L-2300 column oven, and D-2500
chromato-integrator (Hitachi High-Technologies, Tokyo, Japan).
EANa monohydrate (3.00 g, 13.9 mmol) and ␥-cyclodextrin
(7.86 g, 6.06 mmol) were dissolved in 100 ml of 50 mM acetate
buffer (pH 4.0). Toruzyme 3.0 L (5 ml) was added to the solution. The
mixture was stirred for 24 h at 40 ◦C. After checking the progress
of the reaction by HPLC, the temperature of the reaction mixture
was raised to 60 ◦C and 5 ml of amyloglucosidase (1000 units/ml)
was added. Then the resulting mixture was stirred for 2 h at 60 ◦C.
The reaction mixture was chromatographed on a DOWEX 1 × 8 col-
umn (ꢀ 4.6 cm × 24.5 cm, Acetate form, Muromachi Technos, Tokyo,
Japan) eluted with a stepwise gradient of acetic acid–H2O solvent
system (0, 0.1, 0.3, 1 and 3 M). The monoglucoylated erythorbic
acid-containing fraction (3 M acetic acid eluate) was concentrated
to dryness. The resulting residue dissolved in 0.5% formic acid solu-
tion was further chromatographed on a Toyopearl HW-40F column
(ꢀ 4.6 cm × 29 cm, Tosoh, Tokyo, Japan) eluted with 0.5% formic
acid solution. The major glucosylated erythorbic acid-containing
fraction was repeatedly purified by a Toyopearl HW-40F column
(ꢀ 2.5 cm × 95 cm) to yield 2-O-␣-d-glucopyranosyl-d-erythorbic
acid (2.31 g, 49.1%). 1H NMR (600 MHz, D2O-CD3OD (3:1)) ıH: 3.25
(1H, t, J = 9.6 Hz, H-4ꢀ), 3.41 (1H, dd, J = 3.6, 9.6 Hz, H-2ꢀ), 3.50 (2H,
m, H-6), 3.54 (2H, m, H-6ꢀ), 3.61 (1H, t, J = 9.6 Hz, H-3ꢀ), 3.74 (1H,
dd, J = 3.3, 9.6 Hz, H-5ꢀ), 3.93 (1H, m, H-5ꢀ), 4.79 (1H, d, J = 3.0 Hz,
H-4), 5.30 (1H, d, J = 3.6 Hz, H-1ꢀ). 13C NMR (150 MHz, D2O-CD3OD
(3:1)) ıC: 61.1 (C-6ꢀ), 62.4 (C-6), 70.0 (C-4ꢀ), 71.8 (C-5), 72.2 (C-2ꢀ),
73.6 (C-3ꢀ), 74.1 (C-5ꢀ), 78.8 (C-4), 100.0 (C-1ꢀ), 118.8 (C-2), 163.7
2.2. Chemicals
Cyclodextrin glucanotransferase from Thermoanaerobacter sp.
(Toruzyme 3.0 L, batch ACN00261) was kindly provided by
Novozymes (Bagsvaerd, Denmark). Amyloglucosidase solution
from Aspergillus niger (A9913, 4000 units/ml) was purchased from
Sigma Chemical (St. Louis, MO). Erythorbic acid sodium salt (EANa)
monohydrate was from Tokyo Chemical Industry (Tokyo, Japan). ␣-
Cyclodextrin, ␤-cyclodextrin and ␥-cyclodextrin were from Wako
Pure Chemical Industries (Osaka, Japan). 2-O-␣-d-glucopyranosyl-
l-ascorbic acid (AA-2G) was a gift from Hayashibara Biochemical
Laboratories, Inc. (Okayama, Japan). Reagents were used without
further purification. All water used was Milli-Q grade.
2.3. Optimization of reaction conditions for transglycosylation
(C-3), 173.0 (C-1). ESI-HRMS m/z [M−H]−: calcd. for C12H17O11
:
EANa monohydrate (10–100 mg, 46.3–463 ␮mol) and cyclodex-
trin (0.22–0.66 times to the mole of EANa monohydrate) were put
337.0776, found 337.0777. [␣]18 +151.6◦ (c 0.9, H2O).
D

A. Tai et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 19–23
21
20
10
0
10
30
100
EANa monohydrate (mg/ml)
Fig. 2. Effect of substrate concentration on EA monoglucoside formation by CGTase.
The reaction mixture contained EANa monohydrate (10, 30 and 100 mg), ␣-
cyclodextrin and CGTase (50 ␮l) in 1.0 ml of pH 5.5 acetate buffer: ␣-Cyclodextrin
contents of 0.22 equiv. (white bar), 0.44 equiv. (black bar), and 0.66 equiv. (gray bar)
to each mole of EANa monohydrate. The reaction was carried out at 60 ◦C for 24 h.
The reaction mixture was analyzed by HPLC. Values are means + S.D. of triplicate
experiments.
Fig. 3. Effect of reaction pH and temperature on EA monoglucoside formation by
CGTase. The reaction mixture contained EANa monohydrate (30 mg, 139 ␮mol), ␣-
cyclodextrin (58.9 mg, 60.5 ␮mol) and CGTase (50 ␮l) in 1.0 ml of acetate buffer at
pH of 4.0 (black bar), 4.5 (white bar), 5.0 (gray bar) and 5.5 (hatched bar). The reaction
was carried out for 24 h at a range of temperatures from 35 to 60 ◦C. The reaction
mixture was analyzed by HPLC. Values are means + S.D. of triplicate experiments.
3.2. Optimization of EA monoglucoside production
In order to improve the yield of EA monoglucoside, the reaction
of EANa monohydrate (30 mg/ml) and ␣-cyclodextrin (0.44 equiv.)
with CGTase from Thermoanaerobacter sp. was carried out in acetate
buffer (pH 4.0–5.5) at 35–60 ◦C for 24 h (Fig. 3). At each reaction
temperature, the yield tended to increase with decreasing pH. The
tendency became remarkable at 45 ◦C and below. Maximum syn-
thetic yield (31.6%) was observed in pH 4.0 acetate buffer at 40 ◦C.
The yield improved about twice from the initial value of 15.3%
(Fig. 2).
Effects of different substrates on monoglucosylation were
examined under the above-mentioned optimal conditions. ␣-
Cyclodextrin, ␤-cyclodextrin and ␥-cyclodextrin as glucosyl donors
and EA and AA as acceptors were used. With both the use of EA and
AA, the yield decreased with increase in the number of glucose units
of cyclodextrins (Fig. 4). The reaction yields from EA and cyclodex-
trins with CGTase from Thermoanaerobacter sp. were superior to
those from AA and cyclodextrins. These results were contrary to the
results when CGTase from B. stearothermophilus was used [7,22],
suggesting that CGTase from Thermoanaerobacter sp. was suitable
for the monoglucosylation reaction of EA.
3. Results and discussion
3.1. Effect of substrate concentration on EA monoglucoside
formation by CGTase
There are several reports on the production of 2-O-␣-d-
glucopyranosyl-l-ascorbic acid (AA-2G, Fig. 1) by various enzymes
such as ␣-glucosidase, CGTase, amylase, sucrose phosphorylase
and ␣-isomaltosyl glucosaccharide-forming enzyme [24]. CGTase
is considered to be the best enzyme for large-scale production
of AA-2G due to its high level of transglycosylation activity. Aga
et al. reported that CGTase from B. stearothermophilus showed
the highest level of transglycosylation activity when the produc-
tion of AA-2G by CGTases from B. stearothermophilus, B. circulans
and B. macerans was investigated [7]. 2-O-␣-d-glucopyranosyl-d-
erythorbic acid (EA-2G, Fig. 1) was reported to be synthesized
from erythorbic acid (EA) and ␣-cyclodextrin by CGTase from B.
stearothermophilus [22]. However, the yield (ca. 10%) of EA-2G was
much lower than that (ca. 40%) of AA-2G. In preliminary experi-
ments, we confirmed that the yield of EA-2G production by CGTase
from B. macerans was very low compared to that by CGTase from
B. stearothermophilus. We therefore tried to synthesize EA-2G by
using CGTases from other species.
The effect of substrate concentration on EA monoglucoside for-
mation was examined when EA and cyclodextrin were reacted with
a CGTase from Thermoanaerobacter sp. in pH 5.5 acetate buffer
at 60 ◦C for 24 h. Reactions of EANa monohydrate (10, 30, and
100 mg/ml) and ␣-cyclodextrin (0.22–0.66 times to the mole of
EANa monohydrate) were carried out. The type of cyclodextrin and
the reaction pH and temperature were based on the method of
AA-2G synthesis [7]. The yields of EA monoglucoside as a major
product were calculated as AA-2G equivalent. When the concentra-
tion of EANa monohydrate was 30 mg/ml, the reactions generally
gave a high yield (Fig. 2). Maximum synthetic yield (15.3%) was
observed when 0.44 equiv. of ␣-cyclodextrin was used. The yield
exceeded the yield (ca. 10%) of EA-2G production by CGTase from
B. stearothermophilus [22]. When the concentration of EANa mono-
hydrate was 100 mg/ml, the yields were markedly reduced. These
results suggested that EA monoglucoside could be obtained with
high yield by optimization of reaction conditions using CGTase from
Thermoanaerobacter sp.
40
30
20
10
0
α
β
γ
Cyclodextrin
Fig. 4. Effects of different substrates on monoglucosylation by CGTase. The reaction
mixture contained EANa monohydrate (30 mg, 139 ␮mol, black bar) or AANa (30 mg,
151 ␮mol, white bar), cyclodextrin (0.4 equiv.) and CGTase (50 ␮l) in 1.0 ml of pH 4.0
acetate buffer. The reaction was carried out at 40 ◦C for 24 h. The reaction mixture
was analyzed by HPLC. Values are means + S.D. of triplicate experiments.

22
A. Tai et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 19–23
60
A
B
0.4
0.2
50
40
30
20
10
0
2
1
2
1
3
3
4
4
5
6
5
0
C
D
0
2
4
6
8
0.6
2
Time (h)
2
Fig. 6. Hydrolysis of the transglycosylation products by amyloglucosidase. The
reaction mixture of EA and ␣-cyclodextrin with CGTase was treated with (closed
square) or without (open square) amyloglucosidase. The reaction mixture of EA and
␥-cyclodextrin was also treated with (closed circle) or without (open circle) amy-
loglucosidase. The reaction was carried out at 60 ◦C for 8 h. Aliquots were periodically
withdrawn and analyzed by HPLC. Values are means S.D. of triplicate experiments.
Absence of SD bar means that the SD bar is within the symbol.
0.4
0.2
0
1
1
suggesting that it was better for the yield of EA monoglucoside
to use ␥-cyclodextrin when the reaction products are hydrolyzed
with amyloglucosidase. Next, the transglycosylation products of
EA using ␣-cyclodextrin and ␥-cyclodextrin were hydrolyzed by
amyloglucosidase at 60 ◦C for 8 h, and the reaction yields of EA
monoglucoside were determined by HPLC analysis (Fig. 6). Max-
imum yields were observed at 2 h after hydrolysis, and the yields
were gradually decreased by subsequent reaction. The maximum
yield was 47.6% when the transglycosylation products of EA using
␥-cyclodextrin were hydrolyzed. The yield was increased 1.7 times
by the hydrolysis. The maximum yield was 42.7% when the transg-
lycosylation products of EA using ␣-cyclodextrin were hydrolyzed.
It is noteworthy that hydrolysis by amyloglucosidase increased
the yield of EA monoglucoside when ␥-cyclodextrin was used as
a glucosyl donor more than that when ␣-cyclodextrin was used,
although it seemed to be better for the yield of EA monogluco-
side to use ␣-cyclodextrin before the hydrolysis. These results
indicate that additional treatment with amyloglucosidase after
transglycosylation was very effective for high-yield production of
EA monoglucoside.
The effective production process of EA monoglucoside consisted
of two steps: transglycosylation by CGTase from Thermoanaer-
obacter sp. and hydrolysis by amyloglucosidase from A. niger. The
transglycosylation was carried out under optimal reaction condi-
tions: EANa monohydrate concentration of 30 mg/ml, molar ratio
of EA to ␥-cyclodextrin of 1:0.44, CGTase concentration of 50 ␮l/ml,
reaction temperature of 40 ◦C, reaction time of 24 h, and use of pH
4.0 acetate buffer as the reaction solvent. After 24-h reaction, the
temperature of the reaction mixture was raised to 60 ◦C and 50 ␮l
of amyloglucosidase (1000 units/ml) was added per ml of the reac-
tion mixture, and then the reaction mixture was incubated at 60 ◦C
for 2 h. Although changing pH of the buffer solution was needed in
the hydrolysis step in the synthetic method of AA-2G [7], it is an
advantage of this method that change in pH of the buffer solution
is not required in the two steps.
0
10
20
30
0
10
20
30
Time (min)
Time (min)
Fig. 5. HPLC profiles of glycosylation products of EA. EANa monohydrate (30 mg,
139 ␮mol), cyclodextrin (0.44 equiv.) and CGTase (50 ␮l) in 1.0 ml of pH 4.0 acetate
buffer. The reaction was carried out at 40 ◦C for 24 h. Amyloglucosidase was added
to the reaction mixture of CGTase and incubated at 60 ◦C for 2 h. (A) A reaction
mixture of EA and ␣-cyclodextrin with CGTase. (B) A reaction mixture of EA and
␥-cyclodextrin with CGTase. (C) A reaction mixture of EA and ␣-cyclodextrin with
CGTase and the following amyloglucosidase. (D) A reaction mixture of EA and ␥-
cyclodextrin with CGTase and the following amyloglucosidase. Peak 1, EA; Peaks
2–6, glycosylation products of EA.
The HPLC profiles of the reaction mixtures obtained by EA
and ␣-cyclodextrin and by EA and ␥-cyclodextrin are shown in
Fig. 5A and B, respectively. Peak 1 corresponded to EA as a gly-
cosyl acceptor. Peaks 2–6 were transglycosylation products. Peak
2 with the closest retention time to AA-2G, a monoglucoside of
AA, was considered to be a monoglucoside of EA. The amount of
EA monoglucoside produced when ␥-cyclodextrin was used was
less than that when ␣-cyclodextrin was used (Figs. 4 and 5A and
B). However, the types of by-products and the amount of each by-
product increased with the consumption of EA (Fig. 5B) compared
with those when ␣-cyclodextrin was used (Fig. 5A). Peaks 3–6 were
assumed to be EA linked with maltooligosaccharides. AA-2G was
efficiently formed by CGTase using AA and ␣-cyclodextrin [25]. Sev-
eral AA-2-oligoglucosides were also formed in the reaction, and
they could be converted to AA-2G by additional treatment with
amyloglucosidase. Therefore, it seems to be better for the yield of
EA monoglucoside to use ␥-cyclodextrin if the reaction products
are hydrolyzed with an enzyme, although it is better for the yield
to use ␣-cyclodextrin in the reaction with CGTase alone.
To investigate the efficacy of additional hydrolytic treatment
for EA monoglucoside production, the reaction mixtures obtained
by EA and ␣-cyclodextrin and by EA and ␥-cyclodextrin were
treated with amyloglucosidase from A. niger. The contents of EA
monoglucoside markedly increased with disappearance of peaks
3–6 (Fig. 5C and D). Only EA monoglucoside was observed as the
hydrolysate and by-products at trace levels were detected. These
results suggested that the transglycosylation reaction of CGTase
from Thermoanaerobacter sp. had very high regioselectivity. The
intensity of peak 2 in Fig. 5D was higher than that in Fig. 5C,
3.3. Scale-up synthesis and elucidation of the structure of
monoglucosylated EA
The optimal reaction conditions were applied to scale-up
monoglucosylation of EA. EANa monohydrate (3.00 g, 13.9 mmol)
and ␥-cyclodextrin (7.86 g, 6.06 mmol) were dissolved in 100 ml of
acetate buffer (pH 4.0). Toruzyme 3.0 L (5 ml) as a CGTase was added

A. Tai et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 19–23
23
Scheme 1. Synthetic process of EA-2G.
to the solution. The mixture was stirred for 24 h at 40 ◦C. The reac-
Acknowledgments
tion yield was 29.1%, very similar to that in the small-scale reaction
(Fig. 6). The temperature of the reaction mixture was raised to 60 ◦C
and 5 ml of amyloglucosidase (1000 units/ml) was added. Then the
resulting mixture was stirred for 2 h at 60 ◦C. The reaction mixture
was chromatographed on a DOWEX 1 × 8 column and a Toyopearl
HW-40F column. The major glucoylated erythorbic acid-containing
fraction was repeatedly purified by a Toyopearl HW-40F column to
yield the major product (2.31 g, 49.1%). The major product was fully
characterized and identified by the mass spectrum and several NMR
We wish to thank Hayashibara Biochemical Laboratories Inc.,
Okayama, Japan and Novozymes, Bagsvaerd, Denmark for the sup-
ply of AA-2G and Toruzyme 3.0 L, respectively. The authors are
grateful to the SC-NMR Laboratory of Okayama University and the
MS Laboratory of Faculty of Agriculture at Okayama University.
Appendix A. Supplementary data
spectra (1H, 13C, 1H–1H COSY, HSQC, and HMBC). The observed 1
H
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
molcatb.2013.03.011.
and 13C chemical shifts are listed in Experimental section with their
assignment. The HMBC spectrum of the major product showed a
cross peak between H-1ꢀ (ı 5.30) of glucose and C-2 (ı 118.8) of EA,
indicating that the C-2 oxygen of EA was glucosylated. Therefore,
the major product was determined to be 2-O-␣-d-glucopyranosyl-
d-erythorbic acid (EA-2G, Fig. 1). EA-2G was synthesized in 2 steps
in 49.1% overall yield from EA (Scheme 1). These results indicate
that use of the presented method resulted in specific and efficient
formation of EA-2G and suggested that this method can be applied
to mass production of EA-2G.
References
[1] S. Englard, S. Seifter, Annu. Rev. Nutr. 6 (1986) 365–406.
[2] K.A. Naidu, Nutr. J. 2 (2003) 7.
[3] A. Bendich, L.J. Machlin, O. Scandurra, G.W. Burton, D.D.M. Wayner, Adv. Free
Radic. Biol. Med. 2 (1986) 419–444.
[4] B.M. Tolbert, M. Downing, R.W. Carlson, M.K. Knight, E.M. Baker, Ann. N. Y. Acad.
Sci. 258 (1975) 48–69.
[5] I. Yamamoto, N. Muto, E. Nagata, T. Nakamura, Y. Suzuki, Biochim. Biophys.
Acta 1035 (1990) 44–50.
[6] I. Yamamoto, N. Muto, K. Murakami, S. Suga, H. Yamaguchi, Chem. Pharm. Bull.
38 (1990) 3020–3023.
4. Conclusions
[7] H. Aga, M. Yoneyama, S. Sakai, I. Yamamoto, Agric. Biol. Chem. 55 (1991)
1751–1756.
[8] T. Mandai, M. Yoneyama, S. Sakai, N. Muto, I. Yamamoto, Carbohydr. Res. 232
(1992) 197–205.
[9] I. Yamamoto, S. Suga, Y. Mitoh, M. Tanaka, N. Muto, J. Pharmacobio-Dyn. 13
(1990) 688–695.
[10] I. Yamamoto, N. Muto, K. Murakami, J. Akiyama, J. Nutr. 122 (1992) 871–877.
[11] Y. Kumano, T. Sakamoto, M. Egawa, M. Tanaka, I. Yamamoto, Biol. Pharm. Bull.
21 (1998) 662–666.
[12] J.C. Delafuente, R.S. Panush, Int. J. Vitam. Nutr. Res. 50 (1980) 44–51.
[13] B. Leibovitz, B.V. Siegel, Int. J. Vitam. Nutr. Res. 48 (1978) 159–164.
[14] E.J. Goetzl, S.I. Wasserman, I. Gigli, K.F. Austen, J. Clin. Invest. 53 (1974)
813–818.
[15] I. Yamamoto, M. Tanaka, N. Muto, Int. J. Immunopharmacol. 15 (1993) 319–325.
[16] H. Mitsuzumi, M. Kusamiya, T. Kurimoto, I. Yamamoto, Jpn. J. Pharmacol. 78
(1998) 169–179.
[17] M. Tanaka, N. Muto, E. Gohda, I. Yamamoto, Jpn. J. Pharmacol. 66 (1994)
451–456.
[18] K. Maurer, B. Schiedt, Ber. Deut. Chem. Ges. 66 (1933) 1054–1057.
[19] T. Salusjärvi, N. Kalkkinen, A.N. Miasnikov, Appl. Environ. Microbiol. 70 (2004)
5503–5510.
In order to continuously supply EA for long-term cell cultures,
we synthesized a stable EA derivative, EA-2G. In small-scale reac-
tion, the effective production process of EA-2G consisted of two
steps: transglycosylation by CGTase from Thermoanaerobacter sp.
and hydrolysis by amyloglucosidase from A. niger. The transglyco-
sylation was carried out under optimal reaction conditions: EANa
monohydrate concentration of 30 mg/ml, molar ratio of EA to ␥-
cyclodextrin of 1:0.44, CGTase concentration of 50 ␮l/ml, reaction
temperature of 40 ◦C, reaction time of 24 h, and use of pH 4.0 acetate
buffer as the reaction solvent. After 24-h reaction, the temperature
of the reaction mixture was raised to 60 ◦C and 50 ␮l of amyloglu-
cosidase (1000 units/ml) was added per ml of the reaction mixture,
and then the reaction mixture was incubated at 60 ◦C for 2 h. When
the optimal reaction conditions were applied to scale-up synthe-
sis of EA-2G, highly efficient and regioselective formation of EA-2G
was obtained, similar to that observed in the small-scale reaction.
These results suggested that this method can be applied to mass
production of EA-2G. EA-2G, which is much more stable than EA
(Supplementary data), will supply EA to an in vitro cell culture con-
tinuously by enzymatic hydrolysis. We expect EA-2G to be applied
for long-term cell cultures as a useful tool for analyzing the biolog-
ical function of EA.
[20] H.M. Goldman, B.S. Gould, H.N. Munro, Am. J. Clin. Nutr. 34 (1981) 24–33.
[21] M.C. Fidler, L. Davidsson, C. Zeder, R.F. Hurrell, Am. J. Clin. Nutr. 79 (2004)
99–102.
[22] T. Kiryu, H. Murakami, H. Nakano, Y. Okada, M. Senma, Y. Ito, S. Kitahata, Kagaku
to Kogyo (Osaka) (in Japanese) 76 (2002) 443–446.
[23] A. Tai, E. Gohda, J. Chromatogr. B 853 (2007) 214–220.
[24] R. Han, L. Liu, J. Li, G. Du, J. Chen, Appl. Microbiol. Biotechnol. 95 (2012) 313–320.
[25] M. Tanaka, N. Muto, I. Yamamoto, Biochim. Biophys. Acta 1078 (1991) 127–132.