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

Journal of Molecular Catalysis B: Enzymatic 92 (2013) 19–23

Contents lists available at SciVerse ScienceDirect

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 Tai a,∗ , Yuji Iwaoka a , Hideyuki Ito b a b

Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023, Japan 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

Article history: 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

a b s t r a c t In order to continuously supply erythorbic acid (EA) for long-term cell cultures, we synthesized a stable 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 hydrolysis 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. © 2013 Elsevier B.V. All rights reserved.

1. Introduction 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 degradation [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 transglucosylation 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. AA has been reported to enhance immune responses such as lymphocyte proliferation [12], neutrophil function [13] and chemotaxis 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: [email protected] (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

the instability of AA does not permit continuous supply to in vitro cell culture with AA. AA-2G markedly enhanced sheep-red-bloodcell-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 additions 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 suggest that AA-2G is capable of enhancing antibody production in cultured splenocytes via continuous supplementation of AA. Therefore, 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, daraboascorbic 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

20

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 reaction 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]. Separation 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 monoglucosylated products of EA was determined quantitatively as AA-2G equivalent.

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-␣-dglucopyranosyl-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.

2.4. Addition of amyloglucosidase to the glycosylation reaction mixture 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 2.1. General experimental procedure 1 H NMR, 13 C NMR and 2D NMR spectra were recorded on a Varian NMR System 600 MHz instrument. Electron spray ionization (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 analyses 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).

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-glucopyranosyll-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 EANa monohydrate (10–100 mg, 46.3–463 ␮mol) and cyclodextrin (0.22–0.66 times to the mole of EANa monohydrate) were put

2.5. Preparation and purification of 2-O-˛-d-glucopyranosyl-d-erythorbic acid 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 column ( 4.6 cm × 24.5 cm, Acetate form, Muromachi Technos, Tokyo, Japan) eluted with a stepwise gradient of acetic acid–H2 O 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 solution 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%). 1 H NMR (600 MHz, D2 O-CD3 OD (3:1)) ıH : 3.25 (1H, t, J = 9.6 Hz, H-4 ), 3.41 (1H, dd, J = 3.6, 9.6 Hz, H-2 ), 3.50 (2H, m, H-6), 3.54 (2H, m, H-6 ), 3.61 (1H, t, J = 9.6 Hz, H-3 ), 3.74 (1H, dd, J = 3.3, 9.6 Hz, H-5 ), 3.93 (1H, m, H-5 ), 4.79 (1H, d, J = 3.0 Hz, H-4), 5.30 (1H, d, J = 3.6 Hz, H-1 ). 13 C NMR (150 MHz, D2 O-CD3 OD (3:1)) ıC : 61.1 (C-6 ), 62.4 (C-6), 70.0 (C-4 ), 71.8 (C-5), 72.2 (C-2 ), 73.6 (C-3 ), 74.1 (C-5 ), 78.8 (C-4), 100.0 (C-1 ), 118.8 (C-2), 163.7 (C-3), 173.0 (C-1). ESI-HRMS m/z [M−H]− : calcd. for C12 H17 O11 : 337.0776, found 337.0777. [␣]18 D +151.6◦ (c 0.9, H2 O).

A. Tai et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 19–23

21

Reaction yield (%)

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

3.1. Effect of substrate concentration on EA monoglucoside formation by CGTase There are several reports on the production of 2-O-␣-dglucopyranosyl-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 production of AA-2G by CGTases from B. stearothermophilus, B. circulans and B. macerans was investigated [7]. 2-O-␣-d-glucopyranosyl-derythorbic 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 experiments, 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 formation 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 concentration 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 monohydrate 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.

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 synthetic 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 cyclodextrins 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.

40

Reaction yield (%)

3. Results and discussion

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

A

60

B

0.4 2

50

1

2

0.2 3

3 4

4

5

5

6

0

40 30 20 10

C

D

0

0.6

0

2

1 1 0.2

0 10 20 Time (min)

4

6

8

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) amyloglucosidase. 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

Time (h)

2

Intensity (V)

Reaction yield (%)

Intensity (V)

1

30

0

10 20 Time (min)

30

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 glycosyl 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 byproduct 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]. Several 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,

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). Maximum 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 transglycosylation 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 monoglucoside 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 Thermoanaerobacter sp. and hydrolysis by amyloglucosidase from A. niger. The transglycosylation 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 amyloglucosidase (1000 units/ml) was added per ml of the reaction 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. 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 reaction 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 spectra (1 H, 13 C, 1 H–1 H COSY, HSQC, and HMBC). The observed 1 H and 13 C chemical shifts are listed in Experimental section with their assignment. The HMBC spectrum of the major product showed a cross peak between H-1 (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-glucopyranosyld-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. 4. Conclusions In order to continuously supply EA for long-term cell cultures, we synthesized a stable EA derivative, EA-2G. In small-scale reaction, 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 transglycosylation 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 amyloglucosidase (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 synthesis 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 continuously by enzymatic hydrolysis. We expect EA-2G to be applied for long-term cell cultures as a useful tool for analyzing the biological function of EA.

Acknowledgments We wish to thank Hayashibara Biochemical Laboratories Inc., Okayama, Japan and Novozymes, Bagsvaerd, Denmark for the supply 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 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. 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. [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. [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.