One-Pot Synthesis of Hyperoside by a Three ... - ACS Publications

Subscriber access provided by UNIV OF NEWCASTLE

Article

One-pot synthesis of hyperoside by a three-enzyme cascade using a UDP-galactose regeneration system Jianjun Pei, Anna Chen, Linguo Zhao, Fuliang Cao, Gang Ding, and Wei Xiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02320 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on July 4, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

1

One-pot synthesis of hyperoside by a three-enzyme cascade using a

2

UDP-galactose regeneration system

3

Jianjun Peia,b,c#, Anna Chena,b#, Linguo Zhaoa,b,c∗, Fuliang Caoa,b, Gang Dingd, Wei

4

Xiaod*

5

a

6

University, Nanjing, China;

7

b

College of Chemical Engineering, Nanjing Forestry University, Nanjing, China;

8

c

Jiangsu Key Lab for the Chemistry & Utilization of Agricultural and Forest, China

9

d

Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry

Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu Province, China

10 11 12 13 14 15 16 17 18 19

∗ Corresponding author, Linguo Zhao. College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China. Phone: +86-025-85427962. E-mail: [email protected]. Wei

Xiao. Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Haichang South Road, Lianyungang

222001,

China.

Phone:

+86-0518-81152227.

[email protected]. #

These authors contributed equally to this work

1 ACS Paragon Plus Environment

E-mail:


20

Abstract

21

Hyperoside exhibits many biological properties and is more soluble in water than

22

quercetin. A uridine 5ʹ-diphosphate (UDP)-galactose regeneration system and one-pot

23

synthesis of hyperoside was described herein. Glycine max sucrose synthase (GmSUS)

24

was coupled with E. coli UDP-galactose 4-epimerase (GalE) to regenerate

25

UDP-galactose from sucrose and UDP. Petunia hybrida glycosyltransferase (PhUGT)

26

with high activity toward quercetin was used to synthesize hyperoside via the

27

UDP-galactose regeneration system. The important factors for optimal synergistic

28

catalysis were determined. Through the use of a fed-batch operation, the final titer of

29

hyperoside increased to 2134 mg/L, with a corresponding molar conversion of 92%

30

and maximum number of UDP-galactose regeneration cycles (RCmax) of 18.4 under

31

optimal conditions. Therefore, the method described herein for the regeneration of

32

UDP-galactose from UDP and sucrose can be widely used for the glycosylation of

33

flavonoids and other bioactive substances.

34

Keywords: UDP-galactose, hyperoside, glycosyltransferase, sucrose synthase,

35

one-pot synthesis

36 37 38 39 40 41 42 43 44


Page 2 of 30

Page 3 of 30


45

Introduction

46

Flavonoids are a large and structurally diverse group of natural polyphenols found

47

in various plants and play important roles as flower and fruit pigments, UV-B

48

protectants, and signaling molecules, among other roles

49

structures, more than 10,000 flavonoids have been characterized from various plants 3.

50

Their pharmacological activities differ according to their structures. In nature,

51

glycosylated flavonoids are the main derivatives of flavonoids; glycosylation not only

52

improves the solubility and stability of flavonoids but also imparts special activity,

53

improved selectivity, and pharmacological properties to these compounds 4, 5.

1, 2

. Based on their chemical

54

Hyperoside (quercetin 3-O-galactoside), a type of flavonoid-O-glycoside, exhibits

55

higher bioactivities compared to those of quercetin in terms of antiviral,

56

anti-inflammatory

57

Hyperoside is extracted from either Hyperin perforatum L. or the leaves of

58

Zanthoxylum bungeanum via solvent extraction, column chromatography, and

59

crystallization

60

additional secondary metabolites in the extracts, the extraction of hyperoside has

61

proven difficult.

6, 7

, antidepressant

8, 9

, apoptotic

10

, and antifungal

11

effects.

12, 13

. However, given the low concentration and complexity of

62

Glycosyltransferases, which belong to glycosyltransferase family 1, commonly

63

utilize small-molecular-weight compounds as acceptor substrates and uridine

64

5ʹ-diphosphate (UDP)-sugars as donors

65

the glycosylation of quercetin at the 3C-O position by glycosyltransferases in plants.

66

The flavonol 3-O-galactosyltransferase gene (PhUGT) from Petunia hybrida has been

67

cloned and expressed in E. coli

68

gene has been constructed to produce hyperoside

69

growth of recombinant strains and are limited in their ability to permeate the cell

19

5, 14-18

. Hyperoside synthesis occurs through

, and a recombinant strain harboring the PhUGT 14

. However, flavonoids inhibit the



Page 4 of 30

18

70

membrane

71

process requires the consumption of UDP-galactose. Chemical methods applied in the

72

synthesis of UDP-galactose are limited because of side reactions, additional steps,

73

environmental pollution,

74

UDP-galactose regeneration in situ, which can use galactose-1-phosphate and UTP to

75

directly synthesize UDP-galactose by the UDP-sugar pyrophosphorylase (AtUSP)

76

from Arabidopsis

77

enzymes to construct ATP and UTP regenerations for the synthesis of

78

galactose-1-phosphate and UTP.

. Enzymatic methods can overcome these difficulties in vitro, but the

and

low efficiency.

The

enzymatic

method

for

20, 21

, has been reported. However, the system requires additional

79

Sucrose synthase, which catalyzes the reversible conversion of sucrose and UDP

80

into fructose and UDP-glucose, has been used to create a regenerative system of

81

UDP-glucose/UDP

82

interconversion of UDP-galactose and UDP-glucose

83

coupled with GalE to provide a simple and efficient method that can use sucrose as an

84

inexpensive and sustainable carbon source for the synthesis of UDP-galactose. In this

85

paper, a method of synergistic catalysis was established, in which a glycosyl transfer

86

from UDP-galactose to quercetin, catalyzed by recombinant PhUGT, is coupled with

87

the removal of UDP and the regeneration of UDP-galactose catalyzed by recombinant

88

Glycine max sucrose synthase (GmSUS) and recombinant E. coli GalE.

89

Materials and methods

90

Strains, plasmids, media, and chemicals

22-24

.

UDP-galactose

4-epimerase

(GalE)

catalyzes

the

25

. Sucrose synthase can be

91

Escherichia coli strains JM109 and BL21 (DE3) were used for plasmid propagation

92

and recombinant enzyme production. pACYCDuet-1 was purchased from Novagen

93

(Darmstadt, Germany). The strains were grown at 37°C in Luria-Bertani (LB)

94

medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) supplemented with


Page 5 of 30


95

antibiotics when required. UDP, UDP-glucose, and UDP-galactose were obtained

96

from Sigma Chemical Co. (St. Louis, MO, USA).

97

Plasmid construction

98

PhUGT (AAD55985.1), GmSUS (NP_001237525.1), and GalE (NP_415280.3)

99

were synthesized to incorporate E. coli codons. The NcoI site was added to the 5ʹ ends

100

of the genes, the EcoRI site was added to the 3ʹ ends of the genes, and six histidine

101

residues were fused to the C-termini of the recombinant enzymes. The synthesized

102

genes (PhUGT, GmSUS, and GalE) were digested with NcoI and EcoRI and

103

subcloned into the expression vector pACYCDuet-1 at the NcoI and EcoRI sites to

104

create

105

respectively.

106

Purification of recombinant enzymes

pACYCDuet-PhUGT,

pACYCDuet-GmSUS,

and

pACYCDuet-GalE,

107

The pACYCDuet-PhUGT, pACYCDuet-GmSUS, and pACYCDuet-GalE plasmids

108

were transformed into E. coli BL21 (DE3) and induced to express the recombinant

109

enzymes by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final

110

concentration of 0.1 mM at an OD600 of approximately 0.7 using incubation at 20°C

111

for approximately 20 h.

112

Recombinant cells (500 mL) were harvested by centrifugation at 5,000g for 10 min

113

at 4°C; washed twice with distilled water; resuspended in 50 mL of 5 mM imidazole,

114

0.5 mM NaCl, and 20 mM Tris-HCl buffer (pH 7.9); and passed through a French

115

press three times. The cell extracts were then centrifuged (20,000g, 4°C, 30 min). The

116

resulting supernatants were loaded onto an immobilized metal affinity column

117

(Novagen, USA) and eluted with 1 M imidazole, 0.5 M NaCl, and 20 mM Tris-HCl

118

buffer (pH 7.9). The proteins were examined using SDS-PAGE, and the protein bands

119

were analyzed by density scanning with an image analysis system (Bio-Rad, USA).



Page 6 of 30

120

The protein concentrations were determined using the Bradford method with BSA as a

121

standard.

122

Glycosyltransferase activity

123

Glycosyltransferase activity was measured as described previously 19. The reaction

124

mixture, which contained 50 mM phosphate buffer (pH 7.5), 1.25 mM quercetin as a

125

substrate, 2 mM UDP-galactose, and various amounts of PhUGT in 100 µL, was

126

incubated for 10 min at 35°C. The reaction was terminated by adding 400 µL of

127

methanol and then was assayed via high-performance liquid chromatography (HPLC).

128

One unit of enzyme activity was defined as the amount of enzyme necessary to

129

synthesize 1 µmol of hyperoside per min under the assay conditions.

130

The effects of sucrose (0, 10, 50, 100, 200, and 500 mM), fructose (0, 10, 50, 100,

131

and 200 mM), DMSO (1, 5, 10, 15, and 20% v/v), and UDP (0, 0.01, 0.05, 0.1, 0.5, 1,

132

and 5 mM) on the glycosyltransferase activity of PhUGT were determined. The

133

enzyme was incubated with different concentrations of chemical agents for 5 min at

134

35°C before the addition of UDP-galactose to initiate the enzymatic reaction. The

135

activity was determined as described above and was expressed as a percentage of the

136

activity obtained in the absence of the chemical agents.

137

Sucrose synthase activity

138

The sucrose synthase activity was measured as described previously

26

. The BCA

139

assay was used to measure the sucrose synthase activity by detecting the

140

concentration of fructose. The reaction mixture, which contained 50 mM phosphate

141

buffer (pH 7.5), 200 mM sucrose, 1 mM UDP, and various amounts of sucrose

142

synthase in 100 µL, was incubated for 20 min at 35°C. A sample (25 µL) was added to

143

150 µL of assay solution. Afterward, the microtiter plate was covered with plastic foil

144

and incubated for 30 min at 70°C. After cooling to 20°C, the absorbance was


Page 7 of 30


145

measured at 560 nm. One unit of enzyme activity was defined as the amount of

146

enzyme necessary to liberate 1 µmol of fructose per min under the assay conditions.

147

GalE activity

148

The enzyme activity assay was carried out at 35°C in a two-step assay in which the

149

formation of UDP-galactose was coupled to hyperoside production. The reaction

150

mixture, which contained 50 mM phosphate buffer (pH 7.5), 1 mM UDP-glucose, and

151

various amounts of GalE in 100 µL, was incubated for 10 min at 35°C. Then, the

152

mixture was placed in a boiling water bath for 3 min. After cooling to room

153

temperature, a sample (25 µL) was added to 75 µL of reaction mixture, which

154

contained 50 mM phosphate buffer (pH 7.5), 1.5 mM quercetin, and 5 µg of PhUGT,

155

and was incubated for 30 min at 35°C. The reaction was stopped by adding 400 µL of

156

methanol and was then assayed via HPLC. One unit of enzyme activity was defined as

157

the amount of enzyme necessary to synthesize 1 µmol of UDP-galactose per min

158

under the assay conditions.

159

Synergistic catalysis

160

Standard reaction mixtures containing 1.25 mM quercetin, 200 mM sucrose, 0.25

161

mM UDP, 50 mM phosphate buffer (pH 7.5), 5% DMSO (v/v), 100 mU/mL

162

glycosyltransferase, 120 mU/mL sucrose synthase, and 100 mU/mL GalE in 100 µL

163

were incubated for 30 min at 35°C. The reaction was stopped by adding 400 µL of

164

methanol and was then assayed via HPLC.

165

The optimum pH for synergistic catalysis was determined by incubation at 35°C for

166

30 min in 50 mM phosphate buffer from pH 7.0 to 8.0. The optimum temperature for

167

synergistic catalysis was determined in a standard assay ranging from 25 to 45°C in 50

168

mM phosphate buffer, pH 7.5. The optimal ratio among PhUGT, GmSUS, and GalE

169

was determined in a standard assay by adding different ratios of PhUGT, GmSUS, and 7 ACS Paragon Plus Environment


170

GalE. To optimize the conversion conditions, the concentrations of the substrate and

171

DMSO were varied separately: UDP (0–1 mM), sucrose (0–500 mM), quercetin

172

(0.1–5 mM), and DMSO (1–20%, v/v).

173

Fed-batch conversion

174

The quercetin concentrations used in the batch conversions were modified for

175

fed-batch experiments. The reaction solution contained 1.0 mM quercetin, 500 mM

176

sucrose, 0.25 mM UDP, 50 mM phosphate buffer (pH 7.5), 4% DMSO (v/v), 100

177

mU/mL glycosyltransferase, 120 mU/mL sucrose synthase, and 100 mU/mL GalE in 1

178

mL. The reaction was incubated at 35°C and 150 rpm in a thermomixer. Quercetin

179

(1.0 mM) was added from a stock of 200 mM quercetin in DMSO at 0.5, 1, 3, 6, and 9

180

h. Fresh enzymes (100 mU/mL glycosyltransferase, 120 mU/mL sucrose synthase,

181

and 100 mU/mL GalE) were added at 9 h.

182

Product purification

183

The fed-batch reaction solution was harvested by centrifugation at 20,000 g for 10

184

min. The supernatant was applied to a AB-8 column macroporous resin (2.5 x 30 cm,

185

Jianghua, China) equilibrated with the distilled water, and was eluted with 20% and

186

50% ethanol, respectively. The elution with 50% ethanol was collected and

187

evaporated to dryness, and the product was analyzed by HPLC and NMR.

188

HPLC and liquid chromatography-mass spectrometry (LC/MS) analysis

189

HPLC analyses of quercetin and isoquercitrin were performed using an HPLC 1200

190

system (Agilent, USA) and a C18 (250 × 4.6 mm; i.d., 5 µm) column with methanol

191

(A) and distilled water (B) at A/B ratios of 55:45 for 15 min. The flow rate was 0.8

192

mL/min, and detection was performed by monitoring the absorbance at 368 nm.

193

LC/MS for quercetin and hyperoside were analyzed in an LTQ Orbitrap XL LC/MS in

194

negative mode with an ion trap analyzer. The ion spray was operated at 25 Arb N2/min,


Page 8 of 30

Page 9 of 30


195

3.5 kV, and 300°C.

196

Structural identification

197

The structure of the product from synergistic catalysis was determined using the

198

proton and carbon nuclear magnetic resonance (1H-NMR, 13C-NMR) spectrum

199

method (Bruker AVANCE IIII 400) and DMSO-d6 was used as the solvent. 1H NMR

200

(600 MHz, DMSO-d6) δ: ppm 12.64 (s, 1H), 10.86 (s, 1H), 9.73 (s, 1H), 9.16 (s, 1H),

201

7.67 (dd, J = 8.5, 2.2 Hz, 1H), 7.53 (d, J = 2.2 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.41

202

(d, J = 2.0 Hz, 1H), 6.20 (d, J = 2.0 Hz, 1H), 5.38 (d, J = 7.7 Hz, 1H), 5.13 (d, J = 4.2

203

Hz, 1H), 4.86 (d, J = 2.6 Hz, 1H), 4.48 – 4.41 (m, 2H), 3.65 (s, 1H), 3.59 – 3.54 (m,

204

1H), 3.46 (dt, J = 10.6, 5.4 Hz, 1H), 3.34 – 3.32 (m, 1H), 3.31 – 3.27 (m, 1H). 13C

205

NMR (151 MHz, DMSO-d6) δ: 177.52 (s), 164.14 (s), 161.26 (s), 156.29 (d, J = 10.2

206

Hz), 148.49 (s), 144.86 (s), 133.49 (s), 122.04 (s), 121.12 (s), 115.95 (s), 115.20 (s),

207

103.95 (s), 101.78 (s), 98.69 (s), 93.52 (s), 75.87 (s), 73.20 (s), 71.22 (s), 67.94 (s),

208

60.16 (s).

209 210

Results and Discussion

211

Characterization and purification of recombinant enzymes

212

To produce hyperoside by a three-enzyme cascade using a UDP-galactose

213

regeneration system, the recombinant enzymes including PhUGT (AAD55985.1),

214

GmSUS (NP_001237525.1), and GalE (NP_415280.3) were successfully expressed in

215

E. coli 19, 22, 25. Recombinant PhUGT, GmSUS, and GalE in the cell-free extract were

216

purified to gel electrophoretic homogeneity after Ni-NTA affinity chromatography

217

(Fig. 1). To produce hyperoside by synergistic catalysis, it is important for PhUGT to

218

adapt to the reaction conditions of synergistic catalysis so that it can catalyze

219

quercetin with high efficiency, in addition to utilizing UDP-galactose rather than



220

UDP-glucose as the donor. The Kcat/Km value of PhUGT for quercetin was 6.23 x 107

221

M-1 s-1

222

when using UDP-glucose (1 mM) as the donor (Table. S1). The effects of sucrose and

223

fructose on enzyme activity were not significant (Fig. 2a, b), indicating that PhUGT

224

can adapt to the reaction conditions of sucrose synthase. Enzyme activity was

225

enhanced by increasing the concentration of DMSO from 1 to 10% (Fig. 2c). The

226

enzyme activity of PhUGT was gradually inhibited by UDP (Fig. 2d), which suggests

227

that for catalysis, it is important to remove accumulated UDP in situ. Thermostability

228

assays indicated that the residual activity was more than 50% after being incubated at

229

35°C for 3 h. These data showed that recombinant PhUGT represents a potent

230

candidate for the production of hyperoside through synergistic catalysis.

19

. The purified PhUGT cannot catalyze quercetin to produce any product

231

In our previous work, it was found that GmSUS and GalE could tolerate quercetin

232

at concentrations up to 1.5 mM. A large amount of product was generated by PhUGT,

233

GmSUS, and GalE, whereas no product was produced using only PhUGT or PhUGT

234

and GmSUS (Fig. 3a-c). These results show that there is a synergistic reaction among

235

PhUGT, GmSUS, and GalE, in addition to indicating that PhUGT cannot use

236

UDP-glucose as the donor. A comparison of the m/z values of the molecular ion

237

[M-H]- of the enzyme-catalyzed product (463.0886) showed that the differences

238

corresponded to a D-galactose residue in quercetin (301.0355) (Fig. 3d, f), and the

239

product had a retention time similar to the authentic hyperoside (Fig. 3a, c).

240

Furthermore, the 1H-NMR and 13C-NMR spectra were analyzed and compared to

241

reference compounds

242

enzyme-catalyzed product.

243

GmSUS and GalE enhance the efficiency of hyperoside production

244

12

(Fig. S1). These results confirmed that hyperoside was the

GmSUS can be coupled with GalE to produce UDP-galactose from UDP and


Page 10 of 30

Page 11 of 30


245

sucrose. The time-course for UDP-galactose production by GmSUS coupled with

246

GalE is given in Fig. 4. After 20 min of reaction, the UDP-galactose production

247

reached its maximum (0.42 mM), and then more UDP-galactose could not be

248

produced from UDP-glucose, which indicates that the reaction reaches the equilibrium.

249

Thus, the UDP-galactose regeneration system requires a glycosyltransferase to

250

consume the UDP-galactose. However, glycosyltransferase reactions need the

251

UDP-galactose regeneration system to supply the UDP-galactose donor and relieve

252

the end-product inhibition by UDP.

253

To examine the roles of GmSUS and GalE in driving the glycosylation of

254

quercetin, a synergistic reaction was performed to compare the PhUGT conversion of

255

quercetin in the absence and presence of GmSUS and GalE (Fig. 5). The initial rates

256

and final conversion and space-time yields for the production of 1.0 mM hyperoside

257

are summarized in Table 1. Although there was no difference in the initial rates in the

258

absence and presence of GmSUS and GalE with UDP-galactose as donors, the final

259

conversion was significantly lower in the absence of GmSUS and GalE (69.6%) than

260

in their presence (97.6%). These results indicate that it is necessary for PhUGT to

261

remove the accumulated UDP in situ. Using 1.25 mM UDP-galactose instead of 0.25

262

mM UDP in synergistic reactions caused only slight increases in the final conversion

263

(~3.2%) and space-time yield but led to a more significant improvement in the initial

264

rate by approximately 253%. However, in practice, the improvements in the initial

265

rate and similar final conversion do not compensate for the additional cost of

266

replacing UDP with a 5-fold higher concentration of the more expensive

267

UDP-galactose.

268

Optimizing the ratio among PhUGT, GmSUS, and GalE in a one-pot synthesis of

269

hyperoside



270

The effects of the ratio among PhUGT, GmSUS, and GalE on hyperoside

271

production were determined (Table 2). Hyperoside production increased 820% when

272

the amount of PhUGT was increased from 12 to 120 mU/mL, whereas it increased

273

367 and 334% when the amounts of GmSUS and GalE were raised from 12 to 120

274

mU/mL and 10 to 100 mU/mL, respectively. This indicates that the one-pot synthesis

275

of hyperoside catalyzed by PhUGT is the rate-limiting step in the present system.

276

Hyperoside production increased slightly when PhUGT, GmSUS, or GalE continued

277

to increase without changing the amounts of the other two enzymes. Thus, the optimal

278

ratio among PhUGT, GmSUS, and GalE was 120:120:100 (mU/mL).

279

Optimizing the conditions of synergistic catalysis

280

Temperature and pH are important factors in the one-pot synthesis of hyperoside

281

because they affect enzyme-specific activities and stabilities. The results showed that

282

the optimal pH and temperature for synergistic catalysis were pH 7.5 and 40°C,

283

respectively (Fig. 6a, b). However, at 35°C, hyperoside production was 80% of the

284

maximum production, and the lower temperature in general is more conducive to

285

maintaining the thermostabilities of the enzymes. Therefore, a temperature of 35°C

286

was used for the following experiments.

287

Although the poor solubility of quercetin in the reaction system inhibits the activity

288

of PhUGT and the solubility of quercetin can be improved by DMSO, hyperoside

289

production was slightly increased when the concentration of DMSO was increased

290

from 1 to 5%. When the concentration of DMSO exceeded 10%, hyperoside

291

production rapidly decreased (Fig. 6c). Hyperoside production increased with

292

increasing concentration of quercetin, and maximal hyperoside production reached

293

0.46 mM with 1.0 mM quercetin (Fig. 6d). Hyperoside production was rapidly

294

decreased when the concentration of quercetin exceeded 1.5 mM because the activity


Page 12 of 30

Page 13 of 30


295

of GmSUS was inhibited by quercetin in excess of 1.5 mM.

296

Effects of UDP and sucrose on the one-pot synthesis of hyperoside

297

The hyperoside production rate was measured when the initial concentrations of

298

sucrose (10–500 mM; 0.25 mM UDP) or UDP (0.02–1 mM; 200 mM sucrose) were

299

varied, and the results are shown in Fig. 7. The hyperoside production rate was

300

significantly increased as the concentrations of sucrose were increased from 10 to 100

301

mM, and the half-saturation constant of sucrose was 225 mM, which was

302

approximately 8-fold higher than the Km of GmSUS for sucrose 22. UDP has complex

303

effects on synergistic catalysis because it is essential for catalyzing sucrose to

304

UDP-glucose by GmSUS and is a potent inhibitor of the glycosyltransferase activity

305

of PhUGT. The hyperoside production rate was increased approximately 4-fold when

306

the concentration of UDP was increased from 20 to 250 µM and was slightly

307

decreased when the concentration of UDP exceeded 0.5 mM with a half-saturation

308

constant (70 µM) comparable to the Km of GmSUS for UDP. Based on the conversion

309

efficiency, 500 mM sucrose and 0.25 mM UDP were used for the fed-batch

310

experiments.

311

Fed-batch reaction for hyperoside production

312

Based on the results presented above, the optimal conversion conditions were

313

determined and used for hyperoside production. To increase the final concentration of

314

hyperoside in the synergistic reaction and to avoid the inhibition of a high

315

concentration of quercetin on the activity of GmSUS, we changed the operating mode

316

from batch to fed-batch, adding fresh quercetin to a concentration of 1.0 mM once the

317

acceptor substrate had been consumed. Up to 5 rounds of quercetin addition were

318

carried out using a highly concentrated stock solution of 200 mM quercetin in pure

319

DMSO to minimize the resulting volume change. A kinetic analysis of quercetin



320

consumption and hyperoside production over time is shown in Fig. 8. The specific

321

productivity was 459 mg/L/h during the first hour after the initiation of synergistic

322

catalysis. The specific productivity gradually decreased as the reaction proceeded. The

323

specific productivities were 215 mg/L/h over a reaction time of 1-3 h, 107 mg/L/h

324

over a reaction time of 3-9 h, and 67 mg/L/h over a reaction time of 9-18 h. The main

325

cause of the reduction in the specific productivity was the effect of product inhibition.

326

GmSUS is inhibited by fructose with a reported Ki of 9 mM 27. After 18 h, 2134 mg/L

327

hyperoside was produced with a corresponding molar conversion of 92% and

328

maximum number of UDP-galactose regeneration cycles (RCmax) of 18.4 (=4.6/0.25),

329

which was 3.9 times higher than those by the batch reaction (Fig. 5). In general,

330

enzymatic glycosylation is one of the most practical methods for synthesizing a

331

bioactive substrate. However, the high cost of UDP-sugar limits the application of this

332

synthesis approach on a large scale. A multi-enzyme system for UDP-galactose

333

regeneration in situ has been reported. However, the process requires various enzymes

334

to regenerate UDP-galactose over five steps

335

results, the process described herein for UDP-galactose regeneration from sucrose and

336

UDP stands out because of its simplicity. Therefore, UDP-galactose regeneration from

337

UDP and sucrose could be widely used in the glycosylation of flavonoids and other

338

bioactive substances.

20, 21

. Thus, compared to other literature

339 340

Acknowledgments

341

This work was supported by the National Key Research Development Program of

342

China (2016YFD0600805, 2017YFD0600805), the National Natural Science

343

Foundation of China (31570565), the Jiangsu “333” project of cultivation of

344

high-level talents (BRA2015317), the 11th Six Talents Peak Project of Jiangsu


Page 14 of 30

Page 15 of 30


345

Province (2014-JY-011), the Qing Lan Project and the Priority Academic Program

346

Development of Jiangsu Higher Education Institutions (PAPD).

347 348

Supporting Information Available: Relative activity of PhUGT with UDP-galatcose

349

or UDP-glucose. Spectral data for hyperoside.

350 351

REFERENCES

352

(1) Dixon, R. A.; Paiva, N. L., Stress-Induced Phenylpropanoid Metabolism. Plant

353

Cell 1995, 7, 1085-1097.

354

(2) Yonekura-Sakakibara, K.; Tohge, T.; Niida, R.; Saito, K., Identification of a

355

flavonol 7-O-rhamnosyltransferase gene determining flavonoid pattern in Arabidopsis

356

by transcriptome coexpression analysis and reverse genetics. J. Biol. Chem. 2007, 282,

357

14932-14941.

358

(3) Nijveldt, R. J.; van Nood, E.; van Hoorn, D. E.; Boelens, P. G.; van Norren, K.;

359

van Leeuwen, P. A., Flavonoids: a review of probable mechanisms of action and

360

potential applications. Am. J. Clin. Nutr. 2001, 74, 418-425.

361

(4) Wu, X.; Chu, J.; Wu, B.; Zhang, S.; He, B., An efficient novel glycosylation of

362

flavonoid by β-fructosidase resistant to hydrophilic organic solvents. Bioresour.

363

Technol. 2013, 129, 659-662.

364

(5) An, D. G.; Yang, S. M.; Kim, B. G.; Ahn, J. H., Biosynthesis of two quercetin

365

O-diglycosides in Escherichia coli. J. Ind. Microbiol. Biotechnol. 2016, 43, 841-9.

366

(6) Kim, S. J.; Um, J. Y.; Lee, J. Y., Anti-inflammatory activity of hyperoside through

367

the suppression of nuclear factor-kappaB activation in mouse peritoneal macrophages.

368

Am. J. Chin. Med. 2011, 39, 171-181.

369

(7) Comalada, M.; Camuesco, D.; Sierra, S.; Ballester, I.; Xaus, J.; Galvez, J.;



370

Zarzuelo, A., In vivo quercitrin anti-inflammatory effect involves release of quercetin,

371

which inhibits inflammation through down-regulation of the NF-kappaB pathway. Eur.

372

J. Immunol. 2005, 35, 584-592.

373

(8) Zheng, M.; Liu, C.; Pan, F.; Shi, D.; Zhang, Y., Antidepressant-like effect of

374

hyperoside isolated from Apocynum venetum leaves: possible cellular mechanisms.

375

Phytomedicine 2012, 19, 145-149.

376

(9) Butterweck, V.; Jurgenliemk, G.; Nahrstedt, A.; Winterhoff, H., Flavonoids from

377

Hypericum perforatum show antidepressant activity in the forced swimming test.

378

Planta. Med. 2000, 66, 3-6.

379

(10) Cincin, Z. B.; Unlu, M.; Kiran, B.; Bireller, E. S.; Baran, Y.; Cakmakoglu, B.,

380

Apoptotic Effects of Quercitrin on DLD-1 Colon Cancer Cell Line. Pathol. Oncol.

381

Res. 2015, 21, 333-338.

382

(11) Li, S.; Zhang, Z.; Cain, A.; Wang, B.; Long, M.; Taylor, J., Antifungal activity of

383

camptothecin, trifolin, and hyperoside isolated from Camptotheca acuminata. J. Agric.

384

Food Chem. 2005, 53, 32-37.

385

(12) Cao, X.; Wang, Q.; Li, Y.; Bai, G.; Ren, H.; Xu, C.; Ito, Y., Isolation and

386

purification of series bioactive components from Hypericum perforatum L. by

387

counter-current chromatography. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci.

388

2011, 879, 480-488.

389

(13) He, F.; Li, D.; Wang, D.; Deng, M., Extraction and Purification of Quercitrin,

390

Hyperoside, Rutin, and Afzelin from Zanthoxylum bungeanum Maxim Leaves Using

391

an Aqueous Two-Phase System. J. Food Sci. 2016, 81, C1593-C1602.

392

(14) De Bruyn, F.; Van Brempt, M.; Maertens, J.; Van Bellegem, W.; Duchi, D.; De

393

Mey, M., Metabolic engineering of Escherichia coli into a versatile glycosylation

394

platform: production of bio-active quercetin glycosides. Microb. Cell Fact. 2015, 14,


Page 16 of 30

Page 17 of 30


395

138.

396

(15) Kim, B. G.; Kim, H. J.; Ahn, J. H., Production of bioactive flavonol rhamnosides

397

by expression of plant genes in Escherichia coli. J. Agric. Food Chem. 2012, 60,

398

11143-11148.

399

(16) Kim, H. J.; Kim, B. G.; Ahn, J. H., Regioselective synthesis of flavonoid

400

bisglycosides using Escherichia coli harboring two glycosyltransferases. Appl.

401

Microbiol. Biotechnol. 2013, 97, 5275-82.

402

(17) Malla, S.; Pandey, R. P.; Kim, B. G.; Sohng, J. K., Regiospecific modifications of

403

naringenin for astragalin production in Escherichia coli. Biotechnol. Bioeng. 2013,

404

110, 2525-2535.

405

(18) Pei, J.; Dong, P.; Wu, T.; Zhao, L.; Fang, X.; Cao, F.; Tang, F.; Yue, Y., Metabolic

406

engineering of Escherichia coli for astragalin biosynthesis. J. Agric. Food Chem.

407

2016, 64, 7966-7972.

408

(19) Miller, K. D.; Guyon, V.; Evans, J. N.; Shuttleworth, W. A.; Taylor, L. P.,

409

Purification, cloning, and heterologous expression of a catalytically efficient flavonol

410

3-O-galactosyltransferase expressed in the male gametophyte of Petunia hybrida. J.

411

Biol. Chem. 1999, 274, 34011-34019.

412

(20) Yao, Q.; Song, J.; Xia, C.; Zhang, W.; Wang, P. G., Chemoenzymatic syntheses of

413

iGb3 and Gb3. Org. Lett. 2006, 8, 911-914.

414

(21) Tsai, T. I.; Lee, H. Y.; Chang, S. H.; Wang, C. H.; Tu, Y. C.; Lin, Y. C.; Hwang, D.

415

R.; Wu, C. Y.; Wong, C. H., Effective sugar nucleotide regeneration for the large-scale

416

enzymatic synthesis of Globo H and SSEA4. J. Am. Chem. Soc. 2013, 135,

417

14831-14839.

418

(22) Bungaruang, L.; Gutmann, A.; Nidetzky, B., Leloir Glycosyltransferases and

419

natural product glycosylation: biocatalytic synthesis of the C-glucoside nothofagin, a



Page 18 of 30

420

major antioxidant of Redbush Herbal Tea. Adv. Synth. Catal. 2013, 355, 2757-2763.

421

(23) Gutmann, A.; Bungaruang, L.; Weber, H.; Leypold, M.; Breinbauer, R.; Nidetzky,

422

B., Towards the synthesis of glycosylated dihydrochalcone natural products using

423

glycosyltransferase-catalysed cascade reactions. Green Chemistry 2014, 16,

424

4417-4425.

425

(24) Masada, S.; Kawase, Y.; Nagatoshi, M.; Oguchi, Y.; Terasaka, K.; Mizukami, H.,

426

An efficient chemoenzymatic production of small molecule glucosides with in situ

427

UDP-glucose recycling. FEBS. Lett. 2007, 581, 2562-6.

428

(25) Xi, C.; Kowal, P.; Hamad, S.; Fan, H.; Peng, G. W., Cloning, expression and

429

characterization

430

Biotechnology Letters 1999, 21, 1131-1135.

431

(26) Diricks, M.; De Bruyn, F.; Van Daele, P.; Walmagh, M.; Desmet, T.,

432

Identification of sucrose synthase in nonphotosynthetic bacteria and characterization

433

of the recombinant enzymes. Appl. Microbiol. Biotechnol. 2015, 99, 8465-8474.

434

(27) Morell, M.; Copeland, L., Sucrose synthase of soybean nodules. Plant Physiol.

435

1985, 78, 149-154.

of

a

UDP-galactose

4-epimerase

from

436 437 438 439 440 441 442 443 444


Escherichia

coli.

Page 19 of 30


445

Figure legends

446

Fig. 1 SDS-PAGE analysis of recombinant enzymes from E. coli. Lane M: protein

447 448 449

marker; lane 1: purified PhUGT; lane 2: purified GmSUS; lane 3: purified GalE. Fig. 2 Effects of sucrose (a), fructose (b), DMSO (c) and UDP (d) on the glycosyltransferase activity of the purified PhUGT.

450

Fig. 3 HPLC and LC/MS analysis of the products formed from quercetin. (a)

451

Authentic hyperoside. (b) HPLC analysis of the reaction system with PhUGT only. (c)

452

HPLC analysis of the reaction system with PhUGT and GmSUS. (d) HPLC analysis

453

of the reaction system with PhUGT, GmSUS, and GalE. (e/f) LC/MS analysis of the

454

product of the synergistic catalysis.

455

Fig. 4 Time-course for UDP-galactose production by GmSUS coupled with GalE.

456

Fig. 5 PhUGT catalysis of the formation of hyperoside from 1.25 mM

457

UDP-galactose without GmSUS and GalE (diamonds), 1.25 mM UDP-galactose with

458

GmSUS and GalE (squares), and 0.25 mM UDP with GmSUS and GalE (triangles).

459

Fig. 6 Optimization of the bioconversion conditions for hyperoside production by

460

synergistic catalysis. (a) Effects of pH on isoquercitrin production. (b) Effects of

461

temperature on hyperoside production. (c) Effects of DMSO on hyperoside production.

462

(d) Effects of quercetin on hyperoside production.

463 464

Fig. 7 Hyperoside production rate (rp) by synergistic catalysis depends on (a) the sucrose and (b) UDP concentrations.

465

Fig. 8 Hyperoside production in a synergistic reaction involving the feeding of

466

quercetin. Quercetin (1.0 mM) was added at 1, 3, 6, and 9 h. Fresh enzymes (120

467

mU/mL glycosyltransferase, 120 mU/mL sucrose synthase, and 100 mU/mL

468

UDP-galactose 4-epimerase) were added at 9 h.



Page 20 of 30

Table 1. Parameters of the direct and PhUGT-GmSUS-GalE-catalyzed synthesis of hyperoside Conversion Initial production rate of Space-time yield of (%)

hyperoside (mM/h)

hyperoside (mM/h)

mM 69.6

4.3

nd

PhUGT-GmSUS-GalE, 97.6

4.3

2.1

1.7

1.0

PhUGT,

1.25

UDP-galactose

1.25

mM

UDP-galactose PhUGT-GmSUS-GalE, 94.4 0.25 mM UDP nd: less than 1 mM hyperoside


Page 21 of 30


Table 2. Effect of the enzyme concentration on the product yields of hyperosidea Entry

PhUGT

GmSUS

GalE

Hyperoside

Number

(mU/mL)

(mU/mL)

(mU/mL)

(mM)

1

120

120

10

0.103

2

120

120

50

0.265

3

120

120

100

0.321

4

120

120

200

0.345

5

120

12

100

0.094

6

120

60

100

0.172

7

120

240

100

0.445

8

12

120

100

0.042

9

60

120

100

0.212

10

240

120

100

0.408

a: The reaction mixture was incubated for 10 min at 35°C.



Figure 1 43x44mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 22 of 30

Page 23 of 30





Figure 3 138x178mm (300 x 300 DPI)


Page 24 of 30

Page 25 of 30







Page 26 of 30

Page 27 of 30







Page 28 of 30

Page 29 of 30





Abstract graphic 44x23mm (300 x 300 DPI)


Page 30 of 30

One-Pot Synthesis of Hyperoside by a Three ... - ACS Publications

Recommend Documents