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An aqueous-solid system for the highly efficient and environmentally friendly transphosphatidylation catalyzed by phospholipase D to produce phosphatidylserine Binglin Li, Jiao Wang, Xiaoli Zhang, Bin-xia Zhao, and Lu Niu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03448 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on September 29, 2016

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Journal of Agricultural and Food Chemistry

1

An aqueous-solid system for the highly efficient and

2

environmentally friendly transphosphatidylation catalyzed by

3

phospholipase D to produce phosphatidylserine

4 5

Binglin Li, Jiao Wang, Xiaoli Zhang*, Binxia Zhao, Lu Niu

6

Dept. of Chemical Engineering, Northwest University, 229 North Taibai Road,

7

Xi’an, 710000, Shaanxi, China

8 9 10

*Corresponding author: Ph. D. & Prof. Xiaoli Zhang, [email protected]

11 12 13 14 15 16 17 18 19 20 21 22

ACS Paragon Plus Environment


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Abstract

23 24

The

purely

aqueous

system

of

phospholipase

D

(PLD)-mediated

25

transphosphatidylation using pre-existing carriers for the adsorption of

26

phosphatidylcholine (PC) to act as an “artificial interface” was introduced to

27

replace the liquid-liquid system. Toxic organic solvents are avoided during the

28

reaction and the free enzyme can be simply reused by centrifugation. Special

29

attention has been paid to the effect of the pore diameter and surface area of

30

silica gel 60H covered with PC molecules on the yield of phosphatidylserine

31

(PS). Results indicated that the highest PS yield of 99.5 % was achieved.

32

Moreover, 73.6 % of the yield of PS was obtained after being used for 6 batches.

33

This is the first description of the remarkably high reusability of free enzymes

34

for enzymatic synthesis of PS as well. The excellent results make the aqueous-

35

solid system more promising candidates for the industrial production of PS.

36 37

KEYWORDS:

38

Keywords: aqueous-solid system, transphosphatidylation, phosphatidylserine,

39

reusability

40 41 42 43 44


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45


Introduction

46

Phosphatidylserine (PS) has many applications in the food, cosmetic, and

47

pharmaceutical industries.1,2 Early in the last century, Delwaide et al. had

48

reported that the cognitive disorders of the patients with senile dementia was

49

improved by oral administration of bovine brain cortex-derived PS (BC-PS).3

50

Recent clinical studies have also proven that PS plays an important role in

51

revitalizing brain cell membranes and improving memory performance for

52

patients with age-associated memory impairment or Alzheimer’s disease.4–6 In

53

addition, PS is an effective athletic nutrient supplement combating exercise-

54

induced stress and preventing the physiological deterioration via blunting the

55

exercise-induced increase in cortisol levels.7

56

Considering the minor content of PS in the nature, the synthesis of PS

57

becomes more significant. The widely used strategy for producing PS is

58

phospholipase D (PLD)-mediated transphosphatidylation.8,9 Generally, it is

59

carried out in a liquid-liquid system consisting of a water-immiscible organic

60

phase (e.g., diethyl ether, chloroform, toluene), and an aqueous phase.10,11

61

Regarding PS as food or medicine for human use and increasing public health

62

concerns, however, the use of such toxic organic solvents should be avoided.

63

In addition, the health of workers is at stake due to prolonged exposure to the

64

environment containing the volatile organic compounds (VOCs). In light of this,

65

the synthesis of PS in the less toxic solvents has appeared.12,13 A serious

66

drawback of these systems is the complicated process of separation of the



67

product. The expensive price of those less toxic solvents (ionic liquids and γ-

68

valerolactone) is another limiting factor for the industrial production in the

69

coming future. Thus, an ideal choice of the reaction system preformed in the

70

enzymatic synthesis of PS should be a cheap, nontoxic, biocompatible and

71

facile separation system.

72

Water is the best candidate for use as the solvents in pharmaceutical, food,

73

cosmetics and other fields. In this aspect, Dittrich et al. employed the

74

immobilized PLD for the production of phosphatidylglycerol (PG) in an aqueous

75

medium.14 Phosphatidylcholine (PC) was simply dispersed in the aqueous

76

system under stirring. The “effective interface” between PC and PLD is very low

77

thus decreasing the reaction rate and increasing the consumption of enzymes.

78

Afterwards, Yugo Iwasaki et al. reported an aqueous suspension system for

79

PLD-mediated synthesis of PS15. The process for the preparation of powder-

80

adsorbed lecithin was complicated and needed the consumption of energy.

81

Recently, the synthesis was performed using surfactants in the aqueous

82

solution.16 The use of surfactants gives rise to the difficulty of the separation of

83

the product. And the used surfactant, sodium deoxycholate (SDC), is not

84

suitable for food production owing to its toxicity.17

85

In the present work, an aqueous-solid system is introduced for PLD-

86

mediated transphosphatidylation. The surface of carriers was employed as an

87

“artificial interphase” between substrates (PC) and free enzymes (present in the

88

aqueous media). Special attention has been paid to the effect of the pore


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diameter and surface area of carriers covered with PC molecules on the yield

90

of PS. In addition, the aqueous-solid system has been allowed for the reuse of

91

free enzymes. This approach is a promising way to prolong the lifetime of

92

enzymes and reduce the consumption of enzymes. Importantly, it has been

93

successfully applied in the PS synthesis so as to assess its potential in

94

industrial applications.

95

Materials and methods

96

Chemicals and enzymes. Phospholipase D (CAS 9001-87-0 from

97

Streptomyces sp.), phosphatidylcholine (PC), phosphatidylserine (PS) and

98

phosphatidic acid (PA) were purchased from Sigma-Aldrich Co. (St. Louis, MO,

99

USA). PLD was diluted and stored in 0.11 M acetate buffer (3.30×10-4

100

gprotein/mL), pH 5.5, at 4°C; Silica gel 60H was obtained from Qingdao Haiyang

101

Chemical Co., Ltd (China); Cellulose microcrystalline and activated carbon

102

were from Sinopharm Chemical Reagent Co., Ltd (China). Calcium sulfate

103

dehydrate was purchased from Tianjin Beichen Fangzheng Chemical Reagent

104

Factory (China).

105

The adsorption of PC on carrier surfaces. 50 mg PC were dissolved in

106

5 mL of ethyl acetate under ultrasonic vibration. 100 mg silica gel 60H were

107

added into the solution, then 5 mL of acetone or ethanol or ethyl acetate or 5

108

mL of mixture consisting of acetone and water (1:1 v/v) were added. The

109

mixture was incubated at room temperature and 200 rpm for 3 hours. The

110

carrier-adsorbed PC was collected by centrifugation (3500g, 20 min, 15 oC).



111

Samples (10 μL) were taken from the upper liquid to analyze the residual

112

concentration of PC by HPLC.

113

General procedure for enzymatic synthesis of PS. Carrier-adsorbed PC

114

was prepared as above using 100 mg PC and 5 mL acetone. After washing

115

three times with distilled water, the precipitates were resuspended in 10 mL 0.11

116

M acetic acid-sodium acetate buffer (pH 5.5) including 1.31 M L-serine, and 1

117

mL PLD solution was added to react in an incubator at 30°C and 200 rpm for

118

24 hours.

119

After the reaction, the mixture was separated by centrifugation. The

120

precipitates were washed with distilled water (until no enzyme was found in the

121

supernatant solution) and phospholipids adsorbed on carriers were eluted with

122

eluting solvent (chloroform/methanol, 2:1 v/v, 3 mL × 5). Samples (10 μL) were

123

taken from the elution buffer and analyzed by HPLC.

124

The effect of the PC coverage on transphosphatidylation. To

125

investigate the correlation between the yield, the pore diameter and the surface

126

area, the reaction was carried out with different amounts of PC (20, 30, 35, 50,

127

60, 75, 100, 125, 150, 175, 200 mg). The PC coverage (g/g) was defined as the

128

amount of PC adsorbed divided by the amount of silica gel 60H used.

129

Comparison of the reaction system. Transphosphatidylation was carried

130

out in the traditional liquid-liquid system. A mixture consisting of 50 mg PC

131

dissolved in 16 mL of diethyl ether, 7 mL of 1.88 M L-serine solution in 0.11 M

132

acetic acid sodium acetate buffer (pH 5.5), and 1 mL PLD solution, was reacted


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133


in an incubator at 30°C and 200 rpm.

134

Also, the reported aqueous-suspension system was employed.15 50 mg

135

PC and 100 mg calcium sulfate were directly added into 10 mL 0.11 M acetic

136

acid-sodium acetate buffer (pH 5.5) including 1.31 M L-serine, and 1 mL PLD

137

solution was added to react in an incubator at 30°C and 200 rpm for 24 hours.

138

Operational stability of the free enzymes. The operational stabilities of

139

free enzymes in the aqueous-solid system and in the traditional liquid-liquid

140

system during batch reactions were evaluated. After each batch reaction, the

141

free PLD solution was collected and used for the next batch. In each batch

142

reaction, the initial amount of PC is 50 mg.

143

High performance liquid chromatography (HPLC) analysis. The

144

samples were analyzed by a Simadzu LC-20A HPLC (Tokyo, Japan) equipped

145

with a Chromachem evaporative light-scattering detector (ELSD). HPLC

146

separation was on an InertSustain C18 column (5 μm, 4.8 × 150 mm, GL

147

Sciences, Inc.). Mobile phase was acetonitrile/methanol (15:85, v/v) and the

148

flow rate was 1.25 mL/min. The column temperature, nebulizing temperature,

149

and evaporating temperature were controlled at 40, 30, and 40oC, respectively,

150

and the air was used as the nebulizing gas. Each phospholipid was determined

151

by the elution retention time using calibration solutions of phospholipids and

152

concentrations of phospholipids in samples were calculated by the peak area

153

of the integrator.

154

Characterizations of different carriers and carrier-adsorbed PC. A field



155

emission scanning electron microscopy (FE-SEM), (Carl Zeiss SIGMA) was

156

employed to investigate the surface morphology of the hybrid of carrier-

157

adsorbed PC. The accelerated voltage was 5 kV and samples needed coating

158

with gold.

159

A surface area and pore size analyzer (Quantachrome, NOVA 2200e) was

160

employed to measure the surface area and pore size of different samples. The

161

outgas time and outgas temperature of silica gel 60H, calcium sulfate, cellulose

162

microcrystalline, activated carbon were 8, 18, 8, 8 hours and 120, 80, 120,

163

120oC, respectively. The outgas time and the outgas temperature of hybrids of

164

silica gel-adsorbed PC were 18 hours and 50oC. Nitrogen was used in all

165

samples.

166

Results and discussion

167

Transphosphatidylation carried out in the aqueous-solid system. The

168

present work aimed to replace the traditional liquid-liquid system consisting of

169

a toxic organic solvent and an aqueous buffer phase with a purely aqueous

170

system using the pre-existing carrier to adsorb the substrate (PC) to act as an

171

“artificial interface” between PC and enzymes (present in the aqueous solution).

172

The rationale of the aqueous-solid system for transphosphatidylation was a

173

two-step process. Firstly, PC was physically attached on the surface of carriers

174

by adsorption and precipitation. Secondly, carrier-adsorbed PC obtained was

175

used to participate in transphosphatidylation in a purely aqueous solution.

176

Four different systems were examined to optimize the procedure for


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adsorption of PC on carrier surfaces. As can be seen from Figure 1, the PC

178

loading were remarkably influenced by precipitants employed. The maximum

179

value of the PC loading was obtained when acetone was used as the precipitant.

180

To examine the effect of the addition rate of precipitant on the PC loading, a

181

control experiment was carried out to dropwise add the precipitant (acetone).

182

The experimental data indicated that there was no obvious difference in the PC

183

loading, which mainly depended on the amount of precipitant used.

184

In this adsorption system, ethyl acetate and acetone belonging to the class

185

3 residual solvents are promising candidates towards environmentally friendly

186

solvents due to no health-based exposure limit, low toxicity, and solubility in

187

water to assist biodegradation.18 Moreover, they have been rated as Generally

188

Recognized As Safe (GRAS) substances for use as secondary food additives,

189

cosmetic ingredients, synthetic flavoring substances and adjuvants.19,20 In

190

contrast with the traditional liquid-liquid system, the time of the operational

191

process using organic solvents was also decreased, which effectively reduced

192

the possibilities of leaks and spills of organic solvents during the operation.

193

Moreover, the organic solvents were easily collected by centrifugation after

194

each batch, minimizing downstream processing problems.

195

After the adsorption system was confirmed, four different carriers, i.e.,

196

silica gel 60H, calcium sulfate, cellulose microcrystalline and activated carbon,

197

were compared by their performance in the enzymatic synthesis of PS using an

198

aqueous-solid system. Table 1 shows that the carrier employed had an obvious



199

effect on the enzymatic reaction for PS synthesis. The loading amount of PC,

200

the yield of PS and the yield of PA were measured to evaluate the enzymatic

201

reaction.

202

As can be seen from Table 1, the loading amount of PC has no apparent

203

dependence on the carrier used. The adsorption of PC depended on the

204

combination of adsorption and precipitation, but was mainly governed by

205

precipitation. Even if PC did not deposit on the surface of carriers, the

206

precipitant (acetone) would make them self-aggregate and precipitate in

207

solution, leading to no obvious difference of the PC loading in tested carriers.

208

On the other hand, we found that the yield of PS was remarkably influenced

209

by carriers employed. Among these carriers, silica gel 60H showed the best

210

performance. The yield of PS could reach 93.6 %. The yield of PS showed a

211

clear dependence on the specific surface area of carriers. Generally, the

212

specific surface area is one of the most important factors affecting the

213

applications of the carrier in catalysis. It is not hard to imagine that an increase

214

in the specific surface area of carriers can augment the “artificial interphase”

215

between substrates (PC) and free enzymes, and is beneficial to form the

216

monolayer adsorption of PC, thus enhancing catalytic efficiency. Unfortunately,

217

the lowest yield of PS of 0.2 % was recorded for activated carbon which has

218

the largest specific surface area. A reasonable explanation for this phenomenon

219

is that the activated carbon has a very small pore diameter (3.8 nm), which was

220

easily blocked in the process of precipitation of PC resulting in the aggregations


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221

of PC. And the pore diameter of activated carbon was smaller than the

222

dimensions of PLD (7.6 nm × 4.8 nm × 5.7 nm21). The latter could not diffuse

223

into the pore channel of activated carbon to interplay with PC adsorbed on the

224

internal surface of activated carbon.

225

Also, the yield of PS and the yield of PA were significantly influenced by

226

the property of the carrier surface. Activated carbon with a low hydrophilic end

227

group exhibited a poor dispersibility in the aqueous solution, which minimized

228

the contact between carrier-adsorbed PC and PLD, resulting in a low yield. With

229

PC adsorbed on calcium sulfate, the yield of PS (74.7 %) was acceptable, but

230

the formation of PA increased (up to 26.3 %). It may be due to the very high

231

polarity of the calcium sulfate surface. Silica gel 60H with the moderately

232

hydrophilic surface showed the highest yield of PS (93.6 %) and minimized the

233

yield of PA (1.1 %). Moreover, the suitable density (close to water) and silanol

234

groups (Si-OH) existing in the surface of silica gel 60H facilitate the dispersion

235

of silica gel 60H in the aqueous phase and the adsorption of PLD,

236

all contribute to the reduction of diffusional resistance. As we known,

237

transphosphatidylation is kinetically controlled synthesis.26 The affinity between

238

PC and PLD varied with the degree of hydrophilicity/hydrophobicity of the

239

carrier used. It is clear that the yield and the selectivity between

240

transphosphatidylation and hydrolysis varied with the property of the carrier

241

surface.

242

transphosphatidylation catalyzed by different immobilized PLD.14

Similar

phenomenon

has

been

previously


22–25

which

reported

in


243

In the preliminary experiment, we have found that the addition rate of

244

precipitant (acetone) hardly has any effect on the PC loading (Figure 1).

245

However, a rapid precipitant (acetone was added by one time) might result in

246

heterogeneous dispersions of PC on the surface of carriers, which would have

247

an effect on the process of transphosphatidylation. An experiment was carried

248

out with dropwise adding acetone and the result was shown in Table 1. The

249

experiment data indicated that the yield of PS and the yield of PA has no

250

apparent dependence on the addition rate of acetone. It might be explained that

251

silica gel 60H with a large surface area, suitable pore diameter and affinity to

252

organic macromolecules could deal with a rapid adsorption of PC. During the

253

reaction, the carrier-adsorbed PC would have a tendency of parallel movement

254

on the surface of carriers forming a homogeneous PC distribution to decrease

255

free energy of the system.27 Thus, from the practical viewpoint, the acetone was

256

added by one time to simplify the operational process.

257

To examine whether PC was adsorbed on the surface of carriers, the

258

morphology of hybrids of carrier-adsorbed PC was analyzed by scanning

259

electron microscopy (SEM). As shown in Figure 2, aggregations of PC could be

260

clearly observed in almost all Figures except Figure 2a, in which silica gel 60H

261

was selected as the carrier. The edges of silica gel 60H were clear for

262

recognition. It suggested that the distribution of PC on the surface of silica gel

263

60H was more homogeneous compared with others. And silica gel 60H with a

264

smaller size enhanced the collisions between PC and PLD, reduced the


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265

resistance of mass diffusion and increases the catalytic efficiency according to

266

the Stokes-Einstein and collision theory.28,29,30 A hypothesis with respect to the

267

process of transphosphatidylation in the aqueous-solid system was proposed

268

that PC, after bonding with PLD, could be redissolved in solution to further

269

participate in transphosphatidylation, and also, PS was released from PS-PLD

270

complex and adsorbed on the surface of carriers again.

271

More interestingly, even if there were much aggregations of PC, a major

272

portion of the surface of calcium sulfate was unoccupied showing the repulsion

273

between PC and the surface of calcium sulfate (shown in Figure 2b). Therefore,

274

it is unlikely to belong to our proposed aqueous-solid systems, in which carriers

275

are used for the adsorption of PC to create an “artificial interface” between

276

water immiscible substrate (PC) and free enzymes (present in the aqueous

277

media). The theoretical mechanisms behind this observation are not clear yet,

278

which is however beyond the scope of this research.

279

After silica gel 60H was confirmed as the most suitable candidate, the

280

effect of the PC coverage on transphosphatidylation was investigated

281

systematically. And a surface area analyzer was used to analyze the specific

282

surface area and the pore diameter of hybrids of silica gel-adsorbed PC.

283

Results were shown in Figure 3. The curve of the yield of PS could be divided

284

into three parts via the dash line.

285

At low PC coverages, the pore diameters (7.8-9.6nm) were greater than

286

the dimensions of PLD (7.6 nm × 4.8 nm × 5.7 nm21). The latter could freely



287

diffuse into and out of the pore channel of hybrids of silica gel-adsorbed PC. In

288

addition, only a portion of the surface area was occupied; it was hypothesized

289

that a monolayer adsorption of PC might be formed in this stage. Next, the yield

290

of PS showed a slight decline with increasing PC coverages. Due to

291

morphological characteristics of hybrids of silica gel-adsorbed PC viz. the

292

decrease in pore diameters (5.7-7.7 nm), PLD could not freely diffuse into and

293

out of the pore channel and the mass transfer resistance could not be neglected.

294

The multilayer adsorption was inevitable in the second stage. After the surface

295

of carriers was completely occupied, a sharp decrease in the yield of PS was

296

detected. Much PC aggregations were formed, which were even observed by

297

naked eye. During this stage, the portion of pore channel might be completely

298

blocked by PC molecules. PLD could not enter into those pores. The results of

299

multi-point BET (Brunauer-Emmett-Teller) had shown a negative adsorption

300

and the mean pore diameter determined by BJH (Barrett-Joyner-Halenda)

301

method was unstable ranging from 3.1 to 28 nm in triplicate (not shown). A

302

reasonable explanation for the latter might be that the pore channel of silica gel

303

60H was blocked randomly during the process of precipitation. Considering the

304

very high price of PC, any waste of the raw material was unacceptable.

305

Therefore, the appropriate PC coverage should be in the first stage, viz. the PC

306

coverage below 0.5 g/g. The optimal initial amount of PC was determined to be

307

50 mg. The PC coverage was about 0.45 g/g and the yield of PS above 99 %.

308

The transphosphatidylation carried out in the aqueous-solid system was


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309

scaled up from 11 to 110 mL under magnetic stirring to ensure homogeneous

310

mixing for 24 h. The PC loading of 88.2 % (the adsorption procedure was also

311

scaled up from 10 to 100 mL), a 95.4 %yield of PS, and a 1.2 % yield of PA

312

were obtained. These key performance indicators are nearly the same as that

313

obtained in 10 mL scale, which also proved the applicability of using this method

314

for larger scale PS production. In addition, silica gel 60H is a kind of non-toxic

315

and very cheap materials. The increase in the use of it will not raise the cost of

316

production.

317

Comparison of the reaction system. The aqueous-solid system was

318

compared with the traditional liquid-liquid system and the reported aqueous

319

suspension system. The yield of PS and the yield of PA were measured to

320

evaluate the enzymatic reaction.

321

As can be seen from Table 2, the yield of PS in the aqueous-solid system

322

increased dramatically from 61.5 to 99.2 % in comparison with the traditional

323

liquid-liquid system. In the aqueous-solid system, PLD could directly contact

324

with PC due to the creation of an “artificial interphase” between substrates (PC)

325

and free enzymes (existing in the aqueous media) by adsorbing PC onto the

326

surface of silica gel 60H. The mass transfer resistance was reduced effectively

327

and the catalytic efficiency was improved significantly. Moreover, it is worth

328

noting that the hydrolysis of phospholipids was minimized with the use of silica

329

gel 60H as carriers in the aqueous-solid system. The accumulation of amounts

330

of PA was approximately 24 times lower than that in the traditional liquid-liquid



Although

PLD

is

intrinsically

a

hydrolytic

Page 16 of 30

331

system.

enzyme,

332

transphosphatidylation can still proceed well in the aqueous environment under

333

the optimized conditions. Similar phenomenon has been previously reported by

334

several research groups.14,15,31 The theoretical mechanisms behind this

335

observation are not clear yet. A possible explanation is that PLD has different

336

binding sites for water and L-serine molecules.32,33 Thus, the water content may

337

not be the dominant factor affecting the production of PA and is no need to be

338

controlled.

339

The reported aqueous suspension system using calcium sulfate without

340

pre-adsorption15 was compared with our method. The result indicated that the

341

yield of PS (70.2 %) was not ideal in the dilute free enzyme solution. It gave

342

rise to the increase of the hydrolysis of PC, leading to the accumulation of

343

considerable amounts of the undesirable byproduct, PA (27.5 %).

344

Recycling of free enzymes. From a practical viewpoint, the reusability of

345

biocatalyst is one of essential factors to reduce the production cost. Unlike the

346

immobilized enzymes, free enzymes with a highly catalytic activity, however,

347

are more fragile and very difficult for recycling and reuse. If free enzymes can

348

be reused, the economical sustainability will be increased. Encouraged by the

349

excellent experiment results above, the operational stability of free PLD in the

350

aqueous-solid system was investigated and results were presented in Figure 4.

351

Compared with the liquid-liquid system, in which 2.60 % of the yield of PS was

352

obtained in the sixth batch, PLD displayed excellent operational stability in the


Page 17 of 30


353

aqueous-solid system, and 73.6% of the yield of PS was obtained after being

354

used for 6 batches, highlighting the presumable cost-effectiveness of the

355

enzyme. In the traditional liquid-liquid system, the aggregation of PLD might not

356

be avoided due to the use of hydrophobic media, where enzyme is not soluble,

357

leading to a “declined” activity and a poor stability of PLD.34,35 The

358

accumulation of byproduct of choline was another reason for the decline

359

of enzyme activity in repeated reuses, which has the inhibition for PLD-

360

mediated transphosphatidylation.36 The adsorption of PLD on carriers in the

361

aqueous-solid system was also detected. The amount of the adsorbed PLD

362

(mass ratio of the adsorbed PLD to the initial PLD) after each run ranging from

363

2 to 5 % was minimum and could be washed thoroughly with distilled water

364

avoiding the enzyme contamination of the product, meanwhile this

365

phenomenon resulted in a slight decrease in the enzyme activity in the

366

sustainable production. The enzymes in the aqueous-solid system is highly

367

stable, as demonstrated in the 6 days’ reactions during recycling experiment

368

with light loss of the enzyme productivity. The recycling and reuse of the free

369

enzyme can significantly reduce the cost of biocatalyst and, thus, reduce the

370

PS production cost.

371

In general, the enzymatic synthesis of PS is highlighted by the application

372

of the highly efficient and environmentally friendly aqueous-solid system.

373

Byproduct (choline) can be easily removed by centrifugation, which is the

374

contaminant resulting in relatively high production cost. It was also possible to



Page 18 of 30

375

use a kind of food-grade organic solvent (e.g., γ-valerolactone) eluting PS from

376

carriers to thoroughly avoid toxic solvent contamination of the product. In

377

contrast to the reported reaction systems, it is also a more promising way to

378

provide longer lifetime of enzymes for biochemical processes. Thus, the

379

aqueous-solid system for transphosphatidylation shows the great potential for

380

industrial production of PS. Funding Sources

381

This work was supported by grants from Natural Science Basic Research Plan

382

in Shaanxi Province of China (Program No. 2014JM2057). Reference

383

(1)

J.

E.;

Steenbergen,

R.

Metabolism

and

functions

of

phosphatidylserine. Prog. Lipid Res. 2005, 44, 207–234.

384 385

Vance,

(2)

Sakai ., M.; Yamatoya ., H.; Kudo ., S. Pharmacological effects of

386

phosphatidylserine enzymatically synthesized from soybean lecithin on

387

brain functions in rodents. J. Nutr. Sci. Vitaminol. 1996, 42, 47–54.

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Figure captions Figure 1. The effect of different precipitants on the PC loading. Ethyl acetateacetone (dropwise) meant that 5 mL acetone was added dropwise in 1 hour. The data points represent the mean ± SD (error bars) of three independent experiments.

Figure 2. SEM micrographs of hybrids of carrier-adsorbed PC. (a) Silica gel 60H. (b) Calcium sulfate. (c) Cellulose microcrystalline. (d) Activated carbon.

Figure 3. The effect of the PC coverage on transphosphatidylation in the aqueous-solid system. Changes in the yield of PS (black), the pore diameter (red) and the surface area (blue) of hybrids of silica gel 60H-adsorbed PC as a function of the PC coverage (g (PC)/g (carriers)). The dash lines are provided to guide the eye only. The data points represent the mean ± SD (error bars) of three independent experiments.

Figure 4. Investigation of reuse of free PLD. At the end of the reaction, the free PLD was collected and used for the next batch under similar conditions. The yield of PS in each batch (from the first to the sixth batch) was calculated. The data points represent the mean ± SD (error bars) of three independent experiments.


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Table 1 The effect of properties of carriers on the transphosphatidylation Carrier

Specific

Average

The loading

The yield

The yield

surface

pore

amount of

of PS %

of PA %

areaa m2/g

diametera

PC

nm

g (PC)/g

1.10±0.10

93.6±0.8

1.1±0.2

1.05±0.05

94.2±1.0

1.3±0.3

(carriers) Silica gel 60H

246.3±1.7

9.6±0.11

Silica gel 60Hb Calcium sulfate

9.11±0.21

3.8±0.17

1.01±0.12

74.7±3.5

26.3±3.9

Cellulose

1.51±0.07

27.2±0.32

0.85±0.14

15.3±3.1

0.5±0.1

385.3±3.1

3.8±0.08

1.12±0.17

0.20±0.07

0.02±0.01

microcrystalline Activated carbon a

The specific surface area and average pore diameter of various carriers were

determined by a surface area analyzer. bAn experiment was carried out to investigate the effect of the rate of precipitation on the yield of PS. 5 mL acetone was added dropwise in 1hour.



Page 26 of 30

Table 2 Comparison of the reaction system. System

Initial amount

The loading

The yield of

The yield of

of PC mg

amount of PC

PS %

PA %

g (PC)/g (carriers)

Aqueous-solid

54.0±0.2

0.49±0.04

99.2±0.4

0.5±0.3

49.8±0.3

-

70.2±2.5

27.5±3.1

49.5±0.3

-

61.5±2.3

12.1±2.7

system Aqueous suspension systmea Liquid-liquid system


Page 27 of 30


Figure 1

Figure 2



Figure 3


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Figure 4



TOC Graphic


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