Efficient Synthesis of Glucosyl-Î²-Cyclodextrin from Maltodextrins by

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Efficient Synthesis of Glucosyl-#-Cyclodextrin from Maltodextrins by Combined Action of Cyclodextrin Glucosyltransferase and Amyloglucosidase Liuxi Xia, Yuxiang Bai, Wanmeng Mu, Jinpeng Wang, Xueming Xu, and Zhengyu Jin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02079 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017

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Page 1 of 28

Journal of Agricultural and Food Chemistry

Efficient Synthesis

of Glucosyl-β-Cyclodextrin from Maltodextrins

by

Combined Action of Cyclodextrin Glucosyltransferase and Amyloglucosidase

Liuxi Xia†, ‡, #, Yuxiang Bai†, ‡, #, Wanmeng Mu†, Jinpeng Wang†, ‡,Xueming Xu†, ‡, #, Zhengyu Jin*, †, ‡, #

†

State Key laboratory of Food Science and Technology, Jiangnan University, Wuxi

214122, Jiangsu Province, China ‡

School of Food Science and Technology, Jiangnan University, Wuxi 214122,

Jiangsu Province, China #

Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University,

Wuxi 214122, China

* Corresponding author: Email: [email protected] Phone: +86-510-85913660

ACS Paragon Plus Environment


Page 2 of 28

1

Abstract

2

Instead of β-cyclodextrin (β-CD), branched β-CDs have been increasingly used in

3

many aspects as they possess better solubility and higher bioadaptability. But most

4

commercialized

5

glucosyl-β-cyclodextrin (G1-β-CD) prepared via enzymatic approach could be a nice

6

substitute. However, the yield of G1-β-CD was low. Here, we reported a controlled

7

two-step

8

β-cyclodextrin

9

Compared to the single β-CGTase reaction, controlled two-step reaction caused a

10

yield increase of G1-β-CD by 130%. Additionally, the percentage of G1-β-CD was

11

enhanced from 2.4% to 24.0% and the side products α-CD and γ-CD were hydrolyzed

12

because of the coupling activity of β-CGTase. Thus, this controlled two-step reaction

13

might be an efficient approach for industrial production of pure G1-β-CD.

branched

reaction

to

β-CDs

efficiently

glucosyltransferase

were

chemically

prepare

synthesized.

G1-β-CD

(β-CGTase)

and

from

Thus,

maltodextrins

amyloglucosidase

the

by

(AG).

14

15

16

17

Keywords

18

Glucosyl-β-Cyclodextrin,

19

Controlled two-step reaction, Coupling activity.

Cyclodextrin

Glucosyltransferase,

20


Amyloglucosidase,

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21

Introduction

22

β-Cyclodextrin (β-CD) is a native cyclic oligomer composed of seven glucopyranose

23

units linked by α-(1,4) glycosidic bonds. It has an exclusive ability to act as a

24

molecular container for entrapping hydrophobic molecules in their internal cavities.1, 2

25

Thus, it has been widely used in many aspects, including pharmaceutical, cosmetic

26

and food.3 However, the solubility of β-CD is rather low. Branched β-CDs are β-CD

27

derivatives containing mono-/di-saccharides or other functional group which are

28

introduced to the glucosyl units in β-CD via α-(1,6) linkage.4,5 Besides better

29

solubility, they have lower hemolytic activity compared to β-CD.6, 7

30

For the production of branched β-CDs, β-CD pyrolysis at a temperature range

31

between 135 °C to 220 °C has been successfully applied.8,9 Hirsenkorn et al. prepared

32

branched β-CDs in a reaction system containing β-CD and glycosyl donor in a molar

33

ratio from 1:1 to 1:20 in the solvent in the presence of an acid catalyst.10 The currently

34

and

35

hydroxypropyl-β-cyclodextrin

36

synthesized by chemical approaches.11-14

37

Enzymatic synthesis of branched β-CDs has drawn increasing attention and it may

38

replace the chemical way in the future due to its green property. Among all the

39

enzymatic synthesized branched CDs, glucosyl-β-CD (G1-β-CD) is the deepest

40

studied and largest consumed one, in which one glucosyl unit is attached to one –OH

41

group of β-CD via α-(1,6) linkage.15 G1-β-CD has an internal cavity similar to that of

42

β-CD. But it is of much higher aqueous solubility and lower hemolysis. It has been

widely

used

branched and

β-CDs,

such

as

methyl-β-cyclodextrin,

hydroxyethyl-β-cyclodextrin,


are

mainly


Page 4 of 28

43

reported that complex with G1-β-CD can improve the aqueous solubility of puerarin.16

44

G1-β-CD also showed greater affinity for the cationic drugs in comparison with β-CD

45

and G2-β-CD.17 In addition, G1-β-CD could be used as a chiral selector to separate

46

basic drug enantiomers while being applied in capillary zone electrophoresis.18

47

Currently, the G1-β-CD is mainly synthesized by cleavage of the side chain of

48

maltosyl-β-CD (G2-β-CD), by a mixture of taka-amylase and glucoamylase.19 The

49

G2-β-CD is synthesized from β-CD and maltose substrates by a reverse reaction

50

catalyzed by pullulanase. However, the transferase activity of pullulanase is a side

51

activity only if the maltose and CD are both present at a high concentration. And the

52

cost of β-CD and maltose substrate is relatively high. Besides, French et al. have

53

treated starch with an enzyme to produce gelatinized and liquefied starch slurry,

54

followed by treatment with cyclodextrin glucanotransferase.20 But the detailed results

55

have not been reported and the catalysis mechanism is still unclear.

56

Maltodextrins (MDs) are partially hydrolyzed products from starch by ɑ-amylase

57

treatment.21, 22 They contain the original branching α-(1, 6) linkages from starch and

58

possess high aqueous solubility.23 Thus, they may be better substrates for the

59

production of branched CDs compared to starch. In this paper we tried to use the MDs

60

with certain dextrose equivalent (DE) value as a substrate to produce G1-β-CD

61

catalyzed

62

amyloglucosidase (AG).24 The specificities of the used enzymes were characterized.

63

The G1-β-CD product was confirmed by NMR, HPLC and UPLC-MS/MS by

64

comparing with a standard prepared from the laboratory-synthesized and purchased

by

both

Cyclodextrin

Glucosyltransferase


(β-CGTase)

and

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65

G2-β-CD. Moreover, a controlled enzymatic synthesis was tested in this study in order

66

to further improve the yield of G1-β-CD.

67

Materials and methods

68

Materials

69

Cyclodextrin-glycosyltransferase (β-CGTase, EC 2.4.1.19) from Thermoanaerobacter

70

sp. (Toruzyme 3.0 L) was purchased by Novozymes Co. Ltd (Shanghai, China).

71

Amyloglucosidase (AG) from Rhizopus sp. was purchased from Megazyme

72

International Ireland Ltd. (Bray, Ireland). Pullulanase was purchased from Jienengke

73

Biological Engineering Co. Ltd. (Wuxi, China). DE 4-7 maltodextrins and soluble

74

starch were purchased from Sigma-Aldrich Co. LLC. (Shanghai, China). G2-β-CD

75

standard was purchased from Aladdin Biochemical Technology Co. (Shanghai, China).

76

β-CD and maltose were purchased from Sinopharm Group Co. Ltd. (Wuxi, China).

77

All other chemicals were reagent grade.

78

Enzyme purification

79

The purchased β-CGTase was purified by anion-exchange chromatography on an

80

AKTA Pure system (GE Healthcare). A linear gradient of 30 mL with 1 M NaCl in 20

81

mM Tris buffer, pH 8.0, as eluent ran through a 1 mL HiTrap Q HP column (GE

82

Healthcare) at a flow rate of 1 mL/min. Proteins present in the elution peak were

83

collected and then desalted using a 5 mL Hi-Trap desalting column (GE Healthcare)

84

with 20 mM Tris buffer, pH 8.0.

85

Enzyme assays



86

The activity of purified β-CGTase was determined through measuring the yield of

87

β-CD while converting soluble starch. The reaction mixture was composed of 900 µL

88

of 1% (w/v) soluble starch prepared in 20 mM phosphate buffer, pH 5.5 and 100 µL of

89

the enzyme solution.25 The mixture was incubated at 60 °C for 10 min, and then

90

boiled for 15 min to inactivate the enzyme. The generated β-CD was quantified using

91

high performance liquid chromatography described below. One unit of β-CGTase

92

activity was defined as the amount of enzyme used for producing 1 µmol of β-CD per

93

minute under the assay conditions used.

94

The activity of AG was estimated by measuring the release of reducing sugars from

95

soluble starch substrate using the 3, 5-dinitrosalicylic acid (DNS) method.23 The

96

reaction mixture contained 200 µL of gelatinized soluble starch (1%, w/v), 700 µL of

97

20 mM phosphate buffer (pH 6.0), and 100 µL of the enzyme solution. The reaction

98

was implemented at 45 °C for 10 min, followed by boiling for 15 min. Then, one mL

99

distilled water was added and the mixture was incubated at 50 °C for 30 min. After

100

adding 1 mL distilled water, the system was incubated in 50 ° C water bath for 30 min.

101

Then, a volume of 2 mL DNS solution was added. The reaction was carried out for 5

102

min in the boiling water. Afterwards, the reaction system was cooled to room

103

temperature by addition of distilled water to a total volume of 25 mL. Absorbance was

104

measured at 540 nm. One unit of AG activity was defined as the amount of enzyme

105

used for releasing 1 µmol of reducing sugars (expressed as glucose) per minute under

106

assay conditions used.

107

Temperature and pH optimization of enzymes


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108

The optimal temperature and pH of β-CGTase and AG were determined over the pH

109

range of 3.0-9.0 (20 mM sodium acetate buffer, pH 3.0-5.0; 20 mM phosphate buffer,

110

pH 5.5-7.0; 20 mM Tris-HCl buffer, pH 7.5-9.0) at 60 °C and 45 °C, and temperature

111

range of 40-80 °C for CGTase or 25-60 °C for AG at pH 5.5 and 6.0 with 1%

112

maltodextrins (DE 4-7, w/v) as substrate. The activity was measured according to the

113

methods mentioned above.

114

Enzymatic reactions

115

The reaction mixture containing 900 µL of 1% DE 4-7 maltodextrins (20 mM

116

phosphate buffer pH 5.5, w/v), and 100 µL (0.38 U) of the purified β-CGTase solution

117

was incubated at 60 °C for 12 h. Then the mixture was boiled for 15 min. In a parallel

118

experiment, a volume of 100 µL (0.72 U) of the AG solution was added after

119

β-CGTase processing and inactivation. The reaction mixture was then incubated at

120

45 °C for another 12 h, and was boiled for 15 min afterwards.

121

The substrate specificity of AG detected by incubating 1% (w/v) β-CD (20 mM

122

phosphate buffer pH 5.5, w/v) with 0.72 U AG at 45 °C for 12 h.

123

A one-pot conversion of 900 µL of 1% DE 4-7 maltodextrins (20 mM phosphate

124

buffer pH 5.5, w/v) by both 100 µL (0.38 U) of the β-CGTase and 100 µL (0.72 U) of

125

the AG solution were carried out at 45 °C for 12 h followed by boiling for 15 min.

126

In a controlled two-step reaction, one hundred µL (0.38 U) of the purified β-CGTase

127

solution was incubated with 900 µL of 1% DE 4-7 maltodextrins at pH 5.5 and 60 °C

128

for 48 h until the activity of β-CGTase decreased to 0.026 U. Then, an AG solution



129

(100 µL, 0.72 U) was added and the mixture was incubated for another 12 h at 45 °C.

130

Finally, the enzymes were inactivated in boiling water for 15 min.

131

For testing the coupling activity of β-CGTase on α-CD, β-CD, γ-CD and G1-β-CD,

132

one mL mixture solution with α-CD, β-CD, γ-CD and G1-β-CD at the same

133

concentration of 0.4% (w/v) were prepared. Then β-CGTase (0.13 U) and AG (0.24 U)

134

were added. The reaction was carried out at pH 5.5 and 45 °C for 24 h. Every 1 h, 10

135

µL of samples were taken and the reaction was stopped by addition of 10 µL 0.4 M

136

NaOH. Afterwards, all the samples were neutralized by adding 10 µL 0.4 M HCl and

137

analyzed by HPLC.

138

Production of G2-β-CD and G1-β-CD

139

Based on the method published by our group before, the G2-β-CD was synthesized.26

140

Maltose (20.31 g) and β-CD (4.0 g) substrates were catalyzed by purified pullulanase

141

(800 U) at 60 °C for 60 h. Then the mixture was boiled for 15 min to inactivate the

142

enzyme. Then the mixture was treated with 100 µL (0.72 U) AG for 12 h at 45 °C in a

143

shaking water bath and then was boiled for 15 min. The G1-β-CD was further purified

144

by preparative HPLC and was evaluated by LC-MS/MS.

145

Analytical and Preparative High Performance Liquid Chromatography (HPLC)

146

The HPLC equipment consisted of a Shimadzu LC-20AT pump, a Rheodyne injector

147

fitted with a 20 µL loop and a Shimadzu RID-10A detector. Prior to analysis, the

148

reaction samples were filtered through a 0.45 µm syringe filter. Analyses were

149

performed in an APS-2 HYPERSIL column (250 mm×4.6 mm column, Thermo


Page 8 of 28

Page 9 of 28


150

scientific) at 30 °C with a mobile phase of acetonitrile/water (75:25, v/v) at a flow rate

151

of 1 mL/min.

152

In preparative HPLC, the sample was separated by the same procedures described

153

above. A total volume of 300 mL eluent containing pure G1-β-CD was collected and

154

was subjected to rotary evaporation in order to remove the organic solvent. Then, the

155

sample was freeze-dried for further analysis.

156

Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS)

157

The reaction mixtures were analyzed on a Waters Acquity ultra-performance liquid

158

chromatography (UPLC) system coupled to a Waters MALDI SYNAPT Q-TOF mass

159

spectrometer (Waters Co., Milford, MA, USA)，which is equipped with electrospray

160

ionization (ESI) source in both ESI (-) -MS and ESI (-) -MS/MS modes.

161

The reaction samples (1 µL) were separated in Acquity UPLC BEH amide column

162

(2.1 mm×100 mm, 1.7 µm; Waters Co., Miford, MA, USA) with acetonitrile (A) and

163

0.1% ammonia (B) as eluents, at a flow rate of 0.3 mL/min with gradient elution: 0-15

164

min, 80% A and 20% B; 15-17 min, 65% A and 35% B; 15-18 min, 50% A and 50% B.

165

MS parameters were ESI source block temperature of 100 °C, desolvation

166

temperature of 400 °C, capillary voltage of 2.8 kV, and desolvation gas flow of 700

167

L/h. In tandem mass spectrometry mode, the collision gas flow was 50 L/h, and the

168

data was collected from m/z 100 to 3000 Da. The whole system was controlled by

169

MassLynx 4.1 software (Waters Co., Milford, MA, USA).

170

Nuclear Magnetic Resonance (NMR) Spectroscopy



171

The reaction products were desalted and the buffer was exchanged twice with D2O

172

with intermediate lyophilization and then dissolved in 0.5 mL D2O. Resolution

173

enhanced 400 MHz 1D 1H NMR spectra were recorded with a spectral width of 8012

174

Hz in 65 k size of fid (D8 Avance III, Bruker) at a probe temperature of 298 K.

175

Suppression of the HOD signal was achieved by applying a noesygppr 1d pulse

176

sequence. All spectra were processed using MestReNova.

177

Results and discussion

178

Characterization of β-CGTase and AG

179

For characterization of β-CGTase, soluble starch, a regular substrate for starch active

180

enzymes, was used here. As shown in Figure S1A, the optimal reaction pH of purified

181

β-CGTase at 60 °C was 5.5. While determining the optimal pH, two summit points

182

occurred at pH 5.5 and 8.0 (Figure S1B), indicating that the β-CGTase is active under

183

both acidic and alkaline condition. This is similar to other reported β-CGTases from

184

different species.27,28 The product profile demonstrated that the major product was

185

β-CD, while α-CD and γ-CD were generated simultaneously (Figure 1A). The

186

proportions of α-CD，β-CD and γ-CD were 33.3%, 42.2% and 7.3%, respectively.

187

This result showed that the enzyme preferably produces the β-CD. Thus the activity of

188

such β-CGTase was defined based on the productivity of β-CD.

189

For the production of G1-β-CD, AG was also used here in order to cleave the extra

190

glucosyl units from the branching chain of branched-CD synthesized by β-CGTase

191

from maltodextrins. In order to confirm that AG has no cleavage specificity on the CD


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192

part of branched β-CD, β-CD was tested as a single substrate. As shown in Figure 1B,

193

the product profiles before and after processing by AG are identical, demonstrating

194

that AG is not able to process the CD structure. Like β-CGTase, the optimal reaction

195

conditions of AG were determined over a pH range from 3.0 to 9.0 and a temperature

196

range from 25 to 60 °C. As shown in Figure S2, the AG is most active at 45 °C and

197

pH 6.0. While the temperature increased to 55 °C, the activity of AG decreased

198

dramatically by 40%. Especially at the optimal temperature for β-CGTase, approx. 75%

199

of the AG activity was reduced. Thus in one-pot reaction, the temperature should be

200

set below 45 °C.

201

The pH value also strongly influences the activity of AG. For instance, at pH 5.0 and

202

7.0, the activity of AG decreased by approx. 20%. But at pH 5.5 which is the optimal

203

reaction pH for β-CGTase, the activity is as high as the one corresponding to pH 6.0.

204

In conclusion, the pH 5.5 is the best reaction pH condition for one-pot and two-step

205

reactions.

206

Production and identification of standard G1-β-CD from G2-β-CD

207

As reported by our group before, the maltosyl-β-cyclodextrin (G2-β-CD) could be

208

synthesized from maltose and β-CD through the reverse reaction catalyzed by

209

pullulanase.26 Similar activities were also explored in other hydrolases, e.g.

210

galactosidase could catalyze the reverse synthesis of galactosyl-β-CD from galactose

211

and β-CD.29,30 As the G2-β-CD has been successfully prepared and identified before, it

212

was used in this study as a substrate for producing standard G1-β-CD and for testing



213

the cleavage capacity of AG on the side chain of branched β-CD. A product mixture

214

containing maltose, β-CD and G2-β-CD was incubated with AG. After incubation for

215

12 h, the maltose and G2-β-CD disappeared (Figure 2A). Instead, two new peaks

216

emerged. The first peak was confirmed as glucose by comparing with glucose

217

standard. The compound corresponding to the peak X was purified. At the same time,

218

a purchased G2-β-CD was also processed by AG. A peak corresponding to glucose

219

and peak X (Figure 2B) were generated. This also proved that AG is able to

220

hydrolyze the side chains of branched CD but maintain the structure of CD part. The

221

MS/MS result of purified X (Figure 2E) showed that the [M-H] - of peak X is 1295.7,

222

indicating that the molecular weight of the compound corresponding to peak X is

223

1296 which is identical to the molecular weight of G1-β-CD and γ-CD. As the

224

retention time of the peak X is different from that of γ-CD (Figure 2C), peak X most

225

likely represented G1-β-CD.

226

In order to further confirm the structure of the compound corresponding to peak X, a

227

1

228

published by Cui et al. from our group.31 The signal at δ 4.96 ppm belongs to the

229

branching glucose (A1), while the signals around δ 5.08 ppm belong to the glucoses in

230

CD structure including the branched glucose (B1) and the remaining glucoses of the

231

core β-CD.32 The area integration ratio of the peak at δ 5.08 ppm to the one at δ 4.96

232

ppm is 7:1, indicating that the yielded compound may contain one branching glucose

233

and a cyclic maltoheptaose. In addition, the signal around δ 5.40 ppm corresponding

234

to the H-4 in longer branching chain cannot be observed.33 This means that the

H-NMR spectrum was given in Figure 2D. The spectrum was identical with the one


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235

product corresponding to peak X only has one branching glucose and no α-(1, 4)

236

linked linear chain. To conclude, the HPLC, LC-MS/MS and NMR analyses, showed

237

that the generated product corresponding to peak X is G1-β-CD.

238

Production of G1-β-CD from MDs by β-CGTase with or without AG

239

While incubating maltodextrins (DE=4-7) with single β-CGTase, the products are

240

mainly normal CDs (Figure 3A). This is similar to the conversion of soluble starch.

241

The proportions of α-CD ， β-CD and γ-CD were 26.5%, 38.6% and 11.8%,

242

respectively. β-CD was also the leading product and both α-CD and γ-CD occurred.

243

Surprisingly, a peak Y possessing an identical MS/MS spectrum (Figure 3C) and

244

HPLC retention time with G1-β-CD was generated (Figure 3A), demonstrating that

245

the β-CGTase is able to synthesize branched β-CD from highly soluble MDs.

246

However, compared to the normal CDs, the yield of G1-β-CD is relatively low.

247

Except for the G1-β-CD, other long-chain branched-β-CD may be generated. Thus AG

248

which could remove extra glucosyl units of branching chains was used in combination

249

with β-CGTase in order to produce more G1-β-CD. But as shown in Figure 3B, all the

250

compounds were hydrolyzed. This is possibly due to the coupling activity of

251

β-CGTase. When the cyclic structure of CD and branched CD was opened by the

252

coupling activity of β-CGTase, AG immediately hydrolyzed the cleaved chains from

253

their non-reducing ends and resulted in the production of glucose.

254

Controlled two-step reaction for the production of G1-β-CD



255

Thus, here a controlled two-step reaction was used in order to enhance the yield of

256

G1-β-CD. First, the MDs substrate was incubated with β-CGTase for 48 h. After

257

processing, the activity of β-CGTase was reduced from 0.38 U to 0.026 U. Then the

258

product mixture was further processed by AG. The product profile (Figure 4B) is

259

completely different from the one of one-pot reaction. The peak area of normal CDs,

260

especially the α-CD and γ-CD decreased dramatically. But the yield of G1-β-CD was

261

increased by 130% (Table 1). Interestingly, the amount of G1-β-CD is higher than

262

that of α-CD and γ-CD, which are main products in single β-CGTase reaction. The

263

percentage of G1-β-CD increased up to 24.0%, which is almost 10-fold as high as that

264

in single β-CGTase reaction. This is probably because the remaining coupling activity

265

of β-CGTase converted the part of CDs into linear oligosaccharides which were

266

degraded by AG. In addition, the β-CGTase may have low affinity with G1-β-CD, so

267

core ring structure of G1-β-CD is harder to be cleaved compared to normal CDs.

268

In order to explain the phenomenon above, a mimic mixture containing pure α-CD,

269

β-CD, γ-CD and G1-β-CD was processed by a combination of less amount of

270

β-CGTase and AG. As shown in Figure 5A, after 7 h incubation, all substrates were

271

slightly digested. The degradation speed of α-CD and γ-CD was much faster than that

272

of β-CD and G1-β-CD. After 15 h treatment, the normal CDs almost disappeared but

273

G1-β-CD remained. The time-tracking results (Figure 5B) also demonstrated that the

274

rate of the G1-β-CD digestion was much slower than that of normal CDs. After 10 h

275

treatment, more than half of all types of normal CDs were degraded while only 20%

276

of the G1-β-CD was consumed. The coupling activity was determined based on the


Page 14 of 28

Page 15 of 28


277

methods described by Bart A. et al.34 The coupling activities of β-CGTase on α-CD,

278

β-CD, γ-CD and G1-β-CD are 0.34 U, 0.15 U, 0.25 U and 0.06 U, respectively. This

279

confirmed that the β-CGTase preferred to open the core ring structure of regular CDs

280

rather than the G1-β-CD. As separation of the branched β-CD from regular CDs is one

281

of the key problem in product purification, the controlled two-step reaction may be an

282

efficient way for preparing pure G1-β-CD while hydrolyzing the regular CDs.

283

Acknowledgements

284

This work was financially supported by Foundation of self-initiated research project

285

of State Key Laboratory of Food Science and Technology (SKLF-ZZB-201610), the

286

National Natural Science Foundation of China (Grant No. 31230057, 31401524),

287

Jiangnan University Fundamental Research Funds for the Central Universities

288

(JUSRP11706), and Jiangsu Province Science and Technology Support Program

289

(BE2013311).

290

Supporting Information

291

The Supporting Information is available free of charge on the ACS Publications

292

website at http://pubs.acs.org. Temperature and pH profile of the β-CGTase (Figure

293

S1). Temperature and pH profile of the AG (Figure S2).

294

References

295

1. Szejtli, J., Introduction and general overview of cyclodextrin chemistry. Chem. Rev.

296

1998, 98, 1743.

297

2. Takahashi, Y.; Ogawa, T., Total synthesis of cyclodextrins. In Trends in Synthetic



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Carbohydrate Chemistry, American Chemical Society. 1989, 386, 150-158.

299

3. Szente, L.; Fenyvesi, E.; Szejtli, J., Entrapment of iodine with cyclodextrins:

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potential application of cyclodextrins in nuclear waste management. Environ. Sci.

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393

Figure Captions

394

Figure 1. (A) The product specificity of β-CGTase while converting soluble starch as

395

substrate. (B) The substrate specificity of AG while acting on β-CD as substrate.

396

Figure 2. (A) The laboratory synthesized G2-β-CD mixture was treated with AG. (B)

397

The purchased G2-β-CD was treated with AG. (C) Peak X compared with standards of

398

α-CD, β-CD and γ-CD using HPLC. (D) 1H NMR spectrum of peak X. (E) MS/MS

399

spectrum of peak X obtained in the ESI.

400

Figure 3. (A) HPLC spectrum of the products from maltodextrins treated with

401

β-CGTase. (B) HPLC spectrum of the products from maltodextrins treated with

402

β-CGTase and AG. (C) MS/MS spectrum of peak Y in (A) obtained in the ESI.

403

Figure 4. (A) Standards of α-CD, β-CD, γ-CD and G1-β-CD. (B) HPLC of product

404

profile from maltodextrins, treated with controlled two-step reaction in comparison.

405

(C) MS/MS spectrum of G1-β-CD is consistent with standard of G1-β-CD.

406

Figure 5. (A) The HPLC of the product derived from the mixture of α-CD, β-CD,

407

γ-CD and G1-β-CD by β-CGTase and AG process for 0, 7 and 15 h, respectively. (B)

408

The time-tracking consumption of the α-CD, β-CD, γ-CD and G1-β-CD during the

409

process by β-CGTase and AG.



Page 22 of 28

Table Table 1. The concentrations and percentages of α-CD, β-CD, γ-CD and G1-β-CD from maltodextrins, treated with β-CGTase or two-step (β-CGTase + AG) process. α-CD

β-CD

γ-CD

G1-β-CD

2.6

3.9

1.2

0.2

33.6

49.0

15.0

2.4

0.2

1.0

0.2

0.4

9.8

53.4

12.6

24.0

Concentration of products (Maltodextrins+β-CGTase) (mg/mL) Percentage of products (Maltodextrins+β-CGTase) (%, w/w) Concentration of products (Maltodextrins+β-CGTase+A G) (mg/mL) Percentage of products (Maltodextrins+β-CGTase+A G) (%, w/w)


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Figures Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



TOC Graphic


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Efficient Synthesis of Glucosyl-Î²-Cyclodextrin from Maltodextrins by

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