Highly Efficient Enzymatic Acylation of Dihydromyricetin by the

Subscriber access provided by The University of Liverpool

Article

Highly Efficient Enzymatic Acylation of Dihydromyricetin by the Immobilized Lipase with Deep Eutectic Solvents as Co-solvent Shi-Lin Cao, Xiao Deng, Pei Xu, Zi-Xuan Huang, Jian Zhou, Xuehui Li, Minhua Zong, and Wenyong Lou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00011 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 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 25

Journal of Agricultural and Food Chemistry

Highly Efficient Enzymatic Acylation of Dihydromyricetin by the Immobilized Lipase with Deep Eutectic Solvents as Co-solvent Shi-Lin Cao1,4#, Xiao Deng1#, Pei Xu1, Zi-Xuan Huang2, Jian Zhou2, Xue-Hui Li2, Min-Hua Zong1,2,Wen-Yong Lou1,3,*, 1

Lab of Applied Biocatalysis, School of Food Science and Engineering, South China

University of Technology, No. 381 Wushan Road, Guangzhou 510640, China. 2

School of Chemistry and Chemical Engineering, South China University of

Technology, No. 381 Wushan Road, Guangzhou 510640, China. 3

State Key Laboratory of Pulp and Paper Engineering, South China University of

Technology, No. 381 Wushan Road, Guangzhou 510640, China. 4

Department of Food Science, Foshan University, No. 18 Jiangwan Yi Road, Foshan

528000, China. *Corresponding author. Tel.: +86-20-22236669; fax: +86-20-22236669.

1 ACS Paragon Plus Environment


1

Abstract: Novel deep eutectic solvent (DES)-DMSO co-solvent system has been, for

2

the first time, successfully used as the reaction medium for the enzymatic acylation of

3

dihydromyricetin (DMY) catalyzed by the immobilized lipase from Aspergillus niger

4

(ANL). The co-solvent mixture, ChCl:Glycerol-DMSO (1:3, v/v) proved to be the

5

optimal medium. With the newly developed co-solvent, the initial reaction rate of

6

enzymatic acylation of DMY achieved 11.1 mM/h and the conversion of DMY was

7

91.6%. ANL@PD-MNPs is stable and recyclable in this co-solvent, offering 90%

8

conversion rate after repeated use of 5 times. The lipid-solubility of DMY-16-acetate

9

was 10 times higher than that of its raw materials DMY. The results showed that the

10

DMY-16-acetate product exhibits good antioxidative activity. The present research

11

illustrated that the use of DES-DMSO co-solvent may become a feasible alternative

12

for the synthesis of DMY ester.

13

Keywords: dihydromyricetin; deep eutectic solvent; Aspergillus niger lipase;

14

acylation; antioxidant ability

15

16

17

18

19

ACS Paragon Plus Environment

Page 2 of 25

Page 3 of 25


20

INTRODUCTION

21

Dihydromyricetin (DMY), also known as ampelopsin, is a natural flavanonol and

22

shows numerous bioactivities including antioxidant, anti-inflammatory, analgesic,

23

antitussive, antibacterial, anti-thrombotic and anti-tumor activities1-3. However, the

24

highly hydrophilic nature of DMY significantly limits its potential application.

25

Compared with DMY, DMY-acetate was reported to have much higher liposolubility

26

as well as comparable and even improved antioxidative ability4. According to the

27

previous literature, the useful DMY fatty acid esters were industrially synthesized by

28

chemical routes using acid or alkaline catalysts5. But there are a lot of drawbacks with

29

these chemical methods such as unsatisfactory yields, low regioselectivities, harsh

30

reaction conditions as well as time-consuming and arduous purification processes4. In

31

this aspect, the competitive enzyme-catalyzed organic synthesis, for example, using

32

lipase to replace chemical catalysts, has attracted growing interest in the hydrolysis6,

33

epoxidation7, aldol addition8, acylation4, alcoholysis9, because of the excellent

34

regioselectivities, wide substrate specificity, environmentally friendly nature and mild

35

reaction conditions 10. Moreover, the stability, reusability and catalytic performances

36

of enzyme can be generally enhanced through immobilization of enzyme 11.

37

In the enzymatic acylation of DMY, a major problem in choosing solvents is the

38

incompatibility between keeping a high enzyme activity and dissolving substrates

39

well4. This polar substrate is scarcely soluble in enzyme-friendly weak-polar and

40

non-polar solvents such as hexane, while the enzymes are prone to deactivation in

41

high-polar solvents in which the substrates can be scarcely soluble12. Hence, the

42

development of a suitable reaction medium is critical for the enzymatic acylation of 2 ACS Paragon Plus Environment


43

DMY13.

44

Lipase-catalyzed synthesis of DMY fatty acid esters has been carried out in

45

traditional organic solvents including acetonitrile, acetone, tetrahydrofuran (THF),

46

t-butanol, dimethyl sulfoxide (DMSO)14. DMSO is a polar solvent that has the ability

47

to dissolve both polar and nonpolar compounds. Therefore, it is usually used as the

48

solvent or co-solvent for enzymatic synthesis of biodiesel and ester derivatives of

49

polar compounds15. Deep eutectic solvents (DESs) are considered as green solvents9

50

and catalysts16-17. DESs can be prepared in high purity from low-cost starting

51

materials, typically by mixing choline chloride (ChCl) with an hydrogen donor, e.g.

52

an amine, amide, alcohol, or carboxylic acid18. To date, some investigations on

53

enzyme-catalyzed biotransformations in DES-containing systems have been reported,

54

including lipase-catalyzed transesterification19 and alcoholysis9, protease-catalyzed

55

peptide synthesis20-21, epoxide hydrolase-catalyzed asymmetric hydrolysis of

56

1,2-epoxyoctane22. Due to the non-toxicity, biodegradability and low-cost of DES, it

57

is of great interest to investigate the lipase-catalyzed acylation of DMY by using

58

DESs as alternative solvents or co-solvents 19, 23.

59

In this work, we for the first time reported a new and green co-solvent system

60

consisting of DMSO and DES (choline chloride: glycerol) as the reaction medium for

61

the efficient enzymatic acylation of DMY with Aspergillus niger lipase (ANL). In this

62

novel co-solvent system, not only DMY had moderate solubility, but also the

63

immobilized ANL showed much higher catalytic activity than that in either DMSO or

64

DES solvent alone. Besides, the DMY ester product has better lipid solubility and


Page 4 of 25

Page 5 of 25


65

excellent antioxidant activity. Therefore, the newly developed biocatalytic system

66

with the immobilized ANL and the DES-based co-solvent medium is very promising

67

for efficient enzymatic acylation of DMY.

68

EXPERIMENTAL SECTION

69

Materials & Methods

70

Dopamine hydrochloride was purchased from Aladdin (Shanghai, China). Ferric

71

chloride hexahydrate (FeCl3·6H2O) and ferrous chloride tetrahydrate (FeCl2·4H2O)

72

were obtained from Guangzhou Chemical Reagent Co. Ltd. Lipase from Aspergillus

73

niger (2.11 U/g) was from Shenzhen Leveking Bioengineering Co. Ltd., (Shenzhen,

74

China).Vinyl esters used as acyl donors (vinyl acetate, vinyl propionate, vinyl butyrate,

75

vinyl caprylate, vinyl benzoate, vinyl 10-undecenoate, vinyl laurate and vinyl stearate)

76

were purchased from Sigma-Aldrich and TCI Co. Ltd. (Shanghai, China).

77

Dihydromyricetin (>99%) was purchased from Aladdin (Shanghai, China). Other

78

chemicals, purchased from Guangzhou Chemical Reagent Co. Ltd, were of analytical

79

grade unless stated otherwise.

80

Immobilization of Aspergillus niger lipase

81

The ANL (Aspergillus niger lipase) was immobilized according to our previous

82

study24. First, the magnetic nanoparticles (MNPs) were prepared according to the

83

conventional co-precipitation method with some modifications24. 15 mL of Dopamine

84

hydrochloride (2.5 mg/mL) was added to the MNP suspension. The pH of the mixture

85

was adjusted to 8.5 by the addition of NaOH solution (0.1mol/L). After vigorous



86

stirring for 1 h, the polydopamine-coated magnetic nanoparticles (PD-MNPs) were

87

separated by an external magnet and washed three times with deionized water. In

88

order to immobilize ANL, an aqueous solution of ANL (1.5 mg/mL) was prepared

89

first by dissolving the ANL powder in sodium phosphate solution (50 mmol/L,

90

pH=8.0). Then the freshly prepared PD-MNPs solution was added to the ANL

91

solution at 4°C in an ice bath. After stirring at 100 rpm for 12 h, the immobilized ANL

92

(ANL@PD-MNPs) was washed with deionized water and then collected.

93

Synthesis of DESs

94

In this work, three kinds of DESs based on choline chloride were used as the

95

co-solvents in enzymatic acylation of DMY. And three different hydrogen-bond

96

donors (HBDs), such as glycerol, urea and xylitol, were selected for the synthesis of

97

these DESs. A 150-mL jacketed glass vessel and a magnetic stirrer were employed to

98

mix choline chloride with different HBDs in different molar ratios at 80 °C and 300

99

rpm until a homogeneous and colorless liquid formed23. The above-mentioned

100

procedure was performed under nitrogen atmosphere.

101

Enzyme activity assay

102

Lipase activity was assayed as described as followed6: Briefly, the enzyme was

103

added into a solution containing 0.6 mL phosphate buffer (50 mM, pH 8.0) and 0.1

104

mL p-nitrophenyl acetate (80 mM) in 2-propanol. The reaction was carried out at

105

40 °C and 200 rpm for 5 min, and then stopped by adding 5.3 mL ethanol. The

106

mixture was centrifuged at 12,000 g for 5 min (4 °C) and the absorbance of the


Page 6 of 25

Page 7 of 25


107

supernatant at 405 nm was measured. The control experiments were carried out to

108

determine the spontaneous hydrolysis of p-nitrophenyl acetate under the

109

above-mentioned conditions without enzyme 6.

110

Enzymatic acylation of DMY

111

In a typical experiment, 60 U of the ANL@PD-MNPs was mixed with 2 mL pure

112

DES or DMSO-DES co-solvent system. Then, DMY (20 mM) and vinyl acetate (200

113

mM) were added to the above reaction system. The enzymatic acylation of DMY was

114

performed at 40 oC in a shaking water bath (200 rpm). Samples (20 µL) were

115

withdrawn at specified time intervals from the reaction mixture and diluted 20-fold

116

with the methanol solution mobile phase of HPLC prior to HPLC analysis. In order to

117

investigate the effect of the molar ratio of vinyl acetate to DMY, the enzymatic

118

reaction was conducted as follows: 60 U of the ANL@PD-MNPs was mixed with 2

119

mL Ch-Gly/DMSO (1/3, v/v) co-solvent system. Subsequently, DMY (20 mM) and

120

vinyl acetate (50-500 mM) were added, and the reaction was performed at 40 oC in a

121

shaking water bath (200 rpm). For the effect of the enzyme amount, the enzymatic

122

reaction was conducted as follows: 20-70 U of the ANL@PD-MNPs was mixed with

123

2 mL Ch-Gly/DMSO (1/3, v/v) co-solvent system. Then, DMY (20 mM) and vinyl

124

acetate (200 mM) were added, and the reaction was carried out at 40 oC in a shaking

125

water bath (200 rpm). Samples (20 µL) were withdrawn and determined by HPLC

126

according to the above description.

127

RESULTS AND DISSCUSSION



128

The enzymatic acylation of DMY in the ChCl:Glycerol (1:2)(Ch-Gly) and DMSO

129

co-solvent system

130

In the present study, the recovery of lipase activity after immobilization was

131

around 83.6% and the enzyme protein loading of the ANL@PD-MNPs was about 138

132

mg protein /g PD-MNPs carrier. The ANL@PD-MNPs exhibit spherical morphology

133

with the diameter < 30 nm (Figure S1).

134

Reaction media plays an important role in enzymatic reaction. For the enzymatic

135

acylation of DMY, the key is to find a suitable reaction medium that provides

136

appropriate solubility for both the polar DMY and the non-polar acyl donors at the

137

same time. The solubility of DMY in Ch-Gly, Ch-Xylitol and Ch-Urea were 418, 180

138

and 102 mM (Table S1), respectively. The solubility of DMY in Ch-Gly was

139

1.73-fold higher than that in DMSO media (242 mM)4. However, the DMY could not

140

be acylated with vinyl acetate in 100% DESs media. This might be attributed the fact

141

that the non-polar acyl donors vinyl acetate cannot dissolve in the DES media.

142

Moreover, DMSO was a suitable solvent for vinyl acetate. According to the previous

143

research, co-solvent reaction medium consisting of two solvents with different

144

polarity can improve the initial and conversion rate of the enzymatic acylation25-26.

145

Thus, the ANL@PD-MNPs catalytic acylation of DMY in Ch-Gly / DMSO

146

co-solvent with different Ch-Gly volume fraction were performed (Table S2). When

147

the Ch-Gly volume ratio decreased from 3/1 to 1/3, the initial reaction rate (V0)

148

increased from 1.9 to 11.2 mM/h and conversion rate of DMY-16-acetate (CDMY-A)

149

increased from 18.8% to 91.3%. The optimal Ch-Gly / DMSO co-solvent was consist


Page 8 of 25

Page 9 of 25


150

of Ch-Gly/DMSO (1/3, v/v) co-solvent system. This result was superior to the

151

enzymatic reaction by using DMSO as reaction medium (7.5 mM/h and 86.2%),

152

according to our previous literature4.

153

Moreover, the ANL@PD-MNPs catalytic DMY acetylation reaction with higher

154

concentration of DMY (300 mM) in Ch-Gly/DMSO (1/3, v/v) co-solvent system. As

155

shown in Table S3, in the Ch-Gly/DMSO (1/3, v/v) co-solvent system, the V0 and

156

CDMY-A was 55.2 mM/h and 85.3%, respectively, which were significantly higher than

157

that in DMSO (34.9 mM/h and 78.8%). These results might be attributing to the high

158

solubility of DMY in the mixed solvent.

159

In order to investigate the effect of substrate molar ratio between vinyl acetate

160

and DMY on ANL@PD-MNPs catalytic DMY acetylation reaction in Ch-Gly/DMSO

161

(1/3, v/v) co-solvent system, the concentration of DMY was fixed of 20 mM and the

162

concentration of vinyl acetate changed from 50 to 500 mM. As shown in Figure 1, the

163

substrate molar ratio had a significant impact on the initial reaction rate and

164

conversion. The initial reaction rate and conversion rate increased with the increasing

165

substrate molar ratio. When it reached 10:1 (vinyl acetate: DMY), the conversion rate

166

and initial reaction rate were optimal, 91.3% and 11.2 mM/h respectively.

167

The effect of the amount of ANL@PD-MNPs on the ANL@PD-MNPs catalytic

168

DMY acetylation reaction was also investigated. The results were shown in Figure 2,

169

when the enzyme amount was less than 50 U, the initial reaction rate and conversion

170

rate increased rapidly with the increasing amount of enzyme. For example, when the

171

amount of enzyme was 20 U, the initial reaction rate was 4.8 mM/h and the 8 ACS Paragon Plus Environment


172

conversion rate was 70.6%. As the amount enzyme increased to 50 U, the initial

173

reaction rate was 11.1 mM/h and the conversion rate was 91.6% respectively. When

174

the amount of enzyme was more than 50 U, the conversion rate remained the same,

175

the initial reaction rate increased slightly. According to our previous study24, the

176

conversion of the lipase-catalyzed regioselective acylation of DMY was about 79.3%

177

in DMSO. In contrast, a remarkable enhancement in the conversion was observed in

178

the DES/DMSO co-solvent system (91.6%). This demonstrated that the addition of

179

DES into DMSO could significantly improve the enzymatic regioselective acylation

180

of DMY, which was attributable to the following reasons: (1) both the substrate DMY

181

and the examined acylating reagents could be well dissolved in the DES-based

182

co-solvent system; (2) the DES showed good biocompatibility with the enzyme.

183

The operational stability (recycle-ability) of ANL@PD-MNPs was studied in the

184

Ch-Gly/DMSO (1/3, v/v) co-solvent system. As shown in Figure 3, the immobilized

185

lipase ANL@PD-MNPs retained more than 90% of relative activity after successive

186

reuse of 5 cycles, exhibiting excellent recycle-ability. Moreover, ANL@PD-MNPs

187

still kept above 56.7% of relative activity even after being repeatedly used for 10

188

cycles. Certainly, the partial leakage of lipase from the support materials was

189

observed with increasing batch of ANL@PD-MNPs re-use from 5 to 10 cycles,

190

resulting in the gradual decrease of the relative activity. Also, the thermal stability of

191

ANL@PD-MNPs was investigated by incubating ANL@PD-MNPs in the

192

Ch-Gly/DMSO (1/3, v/v) co-solvent system at the operational temperature (40 oC) for

193

24 hour, and it was found that ANL@PD-MNPs maintained more than 98% of its

194

initial activity, indicating the relative good thermal stability under the operational 9 ACS Paragon Plus Environment

Page 10 of 25

Page 11 of 25


195

conditions. Accordingly, the ANL@PD-MNPs-catalyzed acetylation of DMY has a

196

good prospect of industrial application.

197

The lipid-solubility determination of DMY and DMY-16-acetate

198

The solubility of DMY and acetylated products in the oil phase was shown in

199

Table S4. The lipid-solubility of DMY-16-acetate was 0.635g/100g oil, 10 times

200

higher than the solubility of DMY (0.067 g/100g oil).

201

The antioxidant ability of DMY and DMY-16-acetate

202

As shown in Figure 4, the effect of DMY and DMY-16-acetate DPPH free

203

radicals clearance was presented. It showed that DPPH radical scavenging rate of both

204

the DMY and DMY-16-acetate increased constantly during the concentration range of

205

0.25-9 mg/mL. When the concentration of standard control Vc was 4.00 mg/mL,

206

DPPH free radical clearance reached 100%. In terms of IC50 (the concentration of

207

antioxidants when DPPH clearance rate was 50%), DMY and DMY-16-acetate were

208

3.71 mg/mL and 4.16 mg/mL, respectively. Both of them were higher than the IC50 of

209

standard control Vc, 0.51 mg/mL. It is interesting to note that after DMY acetylation,

210

its ability to remove DPPH free radical was improved.

211

Hydroxyl radical has high electronegativity and strong oxidizing ability27. With

212

phenolic hydroxyl group, a strong electron donating group, DMY and their acetylated

213

products can reduce hydroxyl radicals. As shown in Figure 5, with the increase of

214

DMY and DMY-6-acetate concentration, the clearance rate of hydroxyl radical

215

increased accordingly. When the DMY and DMY-16-acetate concentration reached 10 ACS Paragon Plus Environment


216

2.7 mg/mL, hydroxyl radical clearance rate was 94.6% and 98.1%, respectively.

217

Comparing with DMY and DMY-16-acetate, the effect of the Vc on the hydroxyl

218

radicals clearance has a different pattern. When the concentration of Vc was less than

219

1.5 mg/mL, the hydroxyl radical clearance rate increased rapidly. When the

220

concentration of Vc reached 1.5 mg/mL, all of the hydroxyl radicals were basically

221

eliminated. The IC50 of Vc, DMY and DMY-16-acetate were 0.68 mg/mL, 1.73

222

mg/mL and 1.79 mg/mL respectively. This result showed that the hydroxyl radical

223

clearance ability of the product DMY-16-acetate remained stable after the acetylation

224

of DMY. What’s more, DMY and DMY-16-acetate offered better efficiency in

225

removing hydroxyl radicals than DPPH free radical.

226

Fentons reagent, consisting of Fe2+ and H2O2, can oxidize organic compounds to

227

inorganic compounds28. Therefore, it is helpful to reduce the concentration of Fe2+ in

228

Fenton reaction in order to limit the oxidative damage in the human body. Figure 6

229

illustrated the Fe2+-chelating ability of the substrate DMY and the product

230

DMY-16-acetate, with EDTA as the control. As the concentration of EDTA, DMY

231

and DMY-16-acetate increased, their Fe2+-chelating ability increased accordingly.

232

The IC50 of EDTA, DMY and DMY-16-acetate Fe2+ chelating were 0.18 mg/mL, 0.10

233

mg/mL and 0.13 mg/mL respectively. It showed that DMY and DMY-16-acetate

234

exhibited better Fe2+-chelating ability than EDTA. The lower chelating ability of

235

DMY-16-acetate may be attributed to the reduction of its hydroxyl groups after

236

acetylation 29.

237

In this paper, we have demonstrated that the co-solvent mixture of Ch-Gly DES


Page 12 of 25

Page 13 of 25


238

and DMSO is much more suitable for the enzymatic acylation of DMY than the

239

traditional organic solvents. Moreover, this novel co-solvent system can also be used

240

for improving enzymatic synthesis of ester derivatives of polar polyhydroxylated

241

compounds except DMY esters. In the co-solvent, DMSO played an important role in

242

the enhancement of enzymatic acylation by increasing the affinity between enzyme

243

and substrates. Moreover, the Ch-Gly DES, mainly promoted the dissolution of the

244

polar substrate and prevented the inactivation of the enzyme in high concentration of

245

strong-polar organic solution. This co-solvent mixture is apparently superior to the

246

recently reported pure organic reaction media for the enzymatic synthesis of DMY

247

esters, since the use of DMSO usually leads to significant inactivation of the enzyme

248

as well as tedious work-up procedures in the downstream purification. The

249

DMY-16-acetate product exhibits good oxidation resistance, the IC50 of DPPH

250

scavenging, hydroxyl radical scavenging and chelating Fe2+ was 4.16 mg/mL, 1.79

251

mg/mL and 0.13 mg/mL, respectively. Given the above advantages, the novel Ch-Gly

252

DES and DMSO co-solvent developed in this work may find promising application in

253

enzymatic catalysis.

254

ASSOCIATED CONTENT

255

ABBREVIATIONS USED

256

ANL

Aspergillus niger lipase

257

ANL@PD-MNPs

258

ChCl

immobilized Aspergillus niger lipase

choline chloride 12 ACS Paragon Plus Environment


259

DESs

deep eutectic solvents

260

DMSO

dimethyl sulfoxide

261

DMY

dihydromyricetin

262

Gly

glycerol

263

HDBs

hydrogen-bond donors

264

PD-MNPs

265

THF

266

polydopamine-coated magnetic nanoparticles

tetrahydrofuran

ACKNOWLEDGEMENT

267

We wish to thank the Program of State Key Laboratory of Pulp and Paper

268

Engineering (2017ZD05), the National Natural Science Foundation of China

269

(21676104; 21336002; 21376096), the Key Program of Guangdong Natural Science

270

Foundation (S2013020013049), and the Open Funding Project of the State Key

271

Laboratory of Bioreactor Engineering for partially funding this work. We also thank

272

SCUT Doctoral Student Short-Term Overseas Visiting Study Funding Project.

273

SUPPORTING INFORMATION DESCRIPTION

274

The method: HPLC analysis of DMY and DMY-16-acetate; Larger-scale

275

synthesis of DMY esters for the purification and structure determination of DMY

276

esters; The lipid-solubilities and antioxidant abilities of DMY and DMY-16-acetate.


Page 14 of 25

Page 15 of 25


277

Supplementary Tables: Effect of different DESs on solubility of DMY and the

278

conversion of the ANL@PD-MNPs-catalytic acylation of DMY (Table S1). Effect of

279

different Ch-Gly/DMSO volume ratio in Ch-Gly/DMSO cosolvent on acetylation of

280

DMY by ANL@PD-MNPs(Table S2). Enzymatic acetylation of DMY by

281

ANL@PD-MNPs at DMY concentration of 300 mM (Table S3). Solubility of DMY

282

and DMY-16-acetate in oil (Table S4).

283

284

Supplementary Figure: Scanning electron micrograph of the immobilized lipase (Figure S1).



REFERENCE

285 286

(1)

287 288 289

(2)

290 291 292

(3)

293 294 295 296

(4)

297 298 299

(5)

300 301

(6)

302 303 304

(7)

305 306 307

(8)

308 309 310

(9)

311 312 313

(10)

314 315 316

(11)

317 318 319

(12)

320 321 322

(13)

323 324

(14)

325 326

(15)

327 328

(16)

329 330 331

(17)

332 333 334 335

(18)

Shen Y., Lindemeyer A.K., Gonzalez C., Shao X.M., Spigelman I., Olsen R.W., Liang J. Dihydromyricetin as a Novel Anti-Alcohol Intoxication Medication. J. Neurosci., 2012, 32: 390-401 Liu J., Shu Y., Zhang Q.Y., Liu B., Xia J., Qiu M.N., Miao H.L., Li M.Y., Zhu R.Z. Dihydromyricetin Induces Apoptosis and Inhibits Proliferation in Hepatocellular Carcinoma Cells. Oncol. Lett., 2014, 8: 1645-1651 Hou X.L., Tong Q., Wang W.Q., Shi C.Y., Xiong W., Chen J., Liu X., Fang J.G. Suppression of Inflammatory Responses by Dihydromyricetin, a Flavonoid from Ampelopsis Grossedentata, Via Inhibiting the Activation of Nf-Kappa B and Mapk Signaling Pathways. J. Nat. Prod., 2015, 78: 1689-1696 Li W., Wu H., Liu B., Hou X., Wan D., Lou W., Zhao J. Highly Efficient and Regioselective Synthesis of Dihydromyricetin Esters by Immobilized Lipase. J. Biotechnol., 2015, 199: 31-37 Matsumoto T., Tahara S. Ampelopsin, a Major Antifungal Constituent from Salix Sachalinensis, and Its Methyl Ethers. J. Agr. Chem. Soc. Japan, 2001, 75: 659-667 Cao S.L., Huang Y.M., Li X.H., Xu P., Wu H., Li N., Lou W.-Y., Zong M.-H. Preparation and Characterization of Immobilized Lipase from Pseudomonas Cepacia onto Magnetic Cellulose Nanocrystals. Sci. Rep., 2016, 6: 20420 Svedendahl M., Carlqvist P., Branneby C., Allnér O., Frise A., Hult K., Berglund P., Brinck T. Direct Epoxidation in Candida Antarctica Lipase B Studied by Experiment and Theory. ChemBioChem, 2008, 9: 2443-2451 Branneby C., Carlqvist P., Hult K., Brinck T., Berglund P. Aldol Additions with Mutant Lipase: Analysis by Experiments and Theoretical Calculations. J. Molecul. Catal. B: Enzym., 2004, 31: 123-128 Durand E., Lecomte J., Baréa B., Piombo G., Dubreucq E., Villeneuve P. Evaluation of Deep Eutectic Solvents as New Media for Candida Antarctica B Lipase Catalyzed Reactions. Process Biochem., 2012, 47: 2081-2089 Cao S.L., Li X.H., Lou W.Y., Zong M.H. Preparation of a Novel Magnetic Cellulose Nanocrystal and Its Efficient Use for Enzyme Immobilization. J. Mater. Chem. B., 2014, 2: 5522-5530 Cao S.L., Xu P., Ma Y.Z., Yao X.X., Yao Y., Zong M.H., Li X.H., Lou W.Y. Recent Advances in Immobilized Enzymes on Nanocarriers. Chinese J. Catal., 2016, 37: 1814-1823 Yu C.Y., Li X.F., Lou W.Y., Zong M.H. Cross-Linked Enzyme Aggregates of Mung Bean Epoxide Hydrolases: A Highly Active, Stable and Recyclable Biocatalyst for Asymmetric Hydrolysis of Epoxides. J. Biotechnol., 2013, 166: 12-19 Puri M., Barrow C.J., Verma M.L. Enzyme Immobilization on Nanomaterials for Biofuel Production. Trends Biotechnol., 2013, 31: 215-216 Sheldon R.A. Enzyme Immobilization: The Quest for Optimum Performance. Adv. Syn. Catal., 2007, 349: 1289-1307 Ge J., Lu D.A., Wang J., Liu Z. Lipase Nanogel Catalyzed Transesterification in Anhydrous Dimethyl Sulfoxide. Biomacromolecules, 2009, 10: 1612-1618 Tran P.H., Nguyen H.T., Hansen P.E., Le T.N. An Efficient and Green Method for Regioand Chemo-Selective Friedel-Crafts Acylations Using a Deep Eutectic Solvent ([Cholinecl][Zncl2]3). RSC Adv., 2016, 6: 37031-37038 Nguyen H.T., Tran P.H. An Extremely Efficient and Green Method for the Acylation of Secondary Alcohols, Phenols and Naphthols with a Deep Eutectic Solvent as the Catalyst. RSC Adv., 2016, 6: 98365-98368 Smith E.L., Abbott A.P., Ryder K.S. Deep Eutectic Solvents (Dess) and Their Applications. Chem. Rev., 2014, 114: 11060-11082


Page 16 of 25

Page 17 of 25


336

(19)

337 338

(20)

339 340 341

(21)

342 343

(22)

344 345 346 347

(23)

348 349 350

(24)

351 352 353

(25)

354 355 356

(26)

357 358 359

(27)

360 361 362

(28)

363 364 365 366

(29)

Gorke J.T., Srienc F., Kazlauskas R.J. Hydrolase-Catalyzed Biotransformations in Deep Eutectic Solvents. Chem. Comm., 2008, 1235-1237 Cao S.L., Xu H., Li X.H., Lou W.Y., Zong M.H. Papain@Magnetic Nanocrystalline Cellulose Nanobiocatalyst: A Highly Efficient Biocatalyst for Dipeptide Biosynthesis in Deep Eutectic Solvents. ACS Sus. Chem. Eng., 2015, 3: 1589-1599 Maugeri Z., Leitner W., de Maria P.D. Chymotrypsin-Catalyzed Peptide Synthesis in Deep Eutectic Solvents. Eur. J. Org. Chem., 2013, 2013: 4223-4228 Cao S.L., Yue D.M., Li X., Smith T.J., Li N., Zong M.H., Wu H., Ma Y., Lou W.Y. Novel Nano/Micro-Biocatalyst: Soybean Epoxide Hydrolase Immobilized on Uio-66-Nh2 Mof for Efficient Biosynthesis of Enantipure (R)-1, 2-Octanediol in Deep Eutectic Solvents. ACS Sus. Chem. Eng., 2016, 4: 3586-3595 Zhao B.Y., Xu P., Yang F.X., Wu H., Zong M.H., Lou W.Y. Biocompatible Deep Eutectic Solvents Based on Choline Chloride: Characterization and Application to the Extraction of Rutin from Sophora Japonica. ACS Sus. Chem. Eng. 2015, 3: 2746-2755 Deng X., Cao S.-L., Li N., Wu H., Smith T.J., Zong M.-H., Lou W.-Y. A Magnetic Biocatalyst Based on Mussel-Inspired Polydopamine and Its Acylation of Dihydromyricetin. Chinese J. Catal., 2016, 37: 1-2 Ferrer M., Cruces M.A., Bernabe M., Ballesteros A., Plou F.J. Lipase-Catalyzed Regioselective Acylation of Sucrose in Two-Solvent Mixtures. Biotechnol. Bioeng., 1999, 65: 10-16 Adnani A., Basri M., Malek E.A., Salleh A., Rahman M.B.A., Chaibakhsh N., Rahman R. Optimization of Lipase-Catalyzed Synthesis of Xylitol Ester by Taguchi Robust Design Method. Ind. Crop. Prod., 2010, 31: 350-356 Zeng Q.H., Zhang X.W., Xu X.L., Jiang M.H., Xu K.P., Piao J.H., Zhu L., Chen J., Jiang J.G. Antioxidant and Anticomplement Functions of Flavonoids Extracted from Penthorum Chinense Pursh. Food Func., 2013, 4: 1811-1818 Jia S.P., Liang M.M., Guo L.H. Photoelectrochemical Detection of Oxidative DNA Damage Induced by Fenton Reaction with Low Concentration and DNA-Associated Fe2+. J. Phys. Chem. B, 2008, 112: 4461-4464 Klibanov A.M. Why Are Enzymes Less Active in Organic Solvents Than in Water? Trends Biotechnol., 1997, 15: 97-101

367 368 369

370

371

372

373

374



Figure Captions

375

376

Figure 1 Effect of substrate molar ratio on acetylation of DMY by ANL@PD-MNPs.

377

Figure 2 Effect of enzyme dosage on ANL@PD-MNPs-catalyzed acetylation of

378

DMY.

379

Figure 3 Recycling ability of ANL@PD-MNPs in the Ch-Gly/DMSO (1/3, v/v)

380

co-solvent system.

381

Figure 4 Effect of antioxidant concentration on DPPH radical scavenging rate.

382

Figure 5 Effect of antioxidant concentration on hydroxyl radical scavenging rate.

383

Figure 6 Effects of antioxidant concentration on iron-chelating rate.

384


Page 18 of 25

Page 19 of 25


100

12

90 80

10

70

8

60 50

6

Initial reaction rate Yield

4 2

40 30 20

0

5

10

15

20

VA/DMY (mol/mol) Figure 1


25

Conversion(%)

Initial reaction rate (mM/h)

14


100 90

12

80

10

70 60

8

50

6

Initial reaction rate

4 2

Yield

40 30 20

20

30

40

50

60

Enzyme dosage (U) Figure 2


70

Conversion(%)

Initial reaction rate (mM/h)

14

Page 20 of 25


Relative activity (%)

Page 21 of 25

100 90 80 70 60 50 40 30 20 10 0

1

2

3

4

5 6 Batch

7

8

Figure3


9

10

DPPH radical scavenging rate (%)


Page 22 of 25

100 80 60 40 Vitamin C DMY DMY-16-acetate

20 0 0

2

4

6

8

Antioxidant concentration (mg/mL)

Figure4


10


Hydroxyl radical scavenging rate (%)

Page 23 of 25

100 80 60 40 Vitamin C DMY DMY-16-acetate

20 0 0.0

0.5

1.0

1.5

2.0

2.5


Figure 5


3.0


Page 24 of 25

Iron-chelating rate (%)

100 80 60 40 EDTA DMY DMY-16-acetate

20 0 0.0

0.1

0.2

0.3

0.4

0.5


Figure 6


0.6

Page 25 of 25


For the Table of Contents


Highly Efficient Enzymatic Acylation of Dihydromyricetin by the

Recommend Documents