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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
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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
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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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Introduction
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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
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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
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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).
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Samples (10 μL) were taken from the upper liquid to analyze the residual
112
concentration of PC by HPLC.
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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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in an incubator at 30°C and 200 rpm.
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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
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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.
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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
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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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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
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22–25
which
reported
in
Journal of Agricultural and Food Chemistry
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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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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
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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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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
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Although
PLD
is
intrinsically
a
hydrolytic
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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
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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
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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.
388
(3)
Delwaide, P. J.; Gyselynck-Mambourg, A. M.; Hurlet ., A.; Ylieff ., M.
389
Double-blind randomized controlled study of phosphatidylserine in senile
390
demented patients. Acta Neurol. Scand. 1986, 73, 136–140.
391
(4)
Claro, F. T.; Patti, C. L.; Abílio, V. C.; Frussa-Filho, R.; Silva, R. H. Bovine
392
brain phosphatidylserine attenuates scopolamine induced amnesia in
393
mice. Prog. Neuro-Psychopharmacology Biol. Psychiatry 2006, 30, 881–
394
886.
ACS Paragon Plus Environment
Page 19 of 30
395
Journal of Agricultural and Food Chemistry
(5)
Kato-Kataoka, A.; Sakai, M.; Ebina, R.; Nonaka, C.; Asano, T.; Miyamori,
396
T. Soybean-derived phosphatidylserine improves memory function of the
397
elderly Japanese subjects with memory complaints. J. Clin. Biochem.
398
Nutr. 2010, 47, 246–255.
399
(6)
Hirayama, S.; Terasawa, K.; Rabeler, R.; Hirayama, T.; Inoue, T.;
400
Tatsumi, Y.; Purpura, M.; Jäger, R. The effect of phosphatidylserine
401
administration on memory and symptoms of attention-deficit hyperactivity
402
disorder: a randomised, double-blind, placebo-controlled clinical trial. J.
403
Hum. Nutr. Diet. Off. J. Br. Diet. Assoc. 2014, 27, 284–291.
404
(7)
Starks, M. A.; Starks, S. L.; Kingsley, M.; Purpura, M.; Jäger, R. The
405
effects of phosphatidylserine on endocrine response to moderate
406
intensity exercise. J. Int. Soc. Sports Nutr. 2008, 5, 1–6.
407
(8)
Hatanaka, T.; Negishi, T.; Kubota-Akizawa, M.; Hagishita, T. Purification,
408
characterization, cloning and sequencing of phospholipase D from
409
Streptomyces septatus TH-2. Enzym. Microb. Technol. 2002, 31, 233–
410
241.
411
(9)
Ogino, C.; Negi, Y.; Matsumiya, T.; Nakaoka, K.; Kondo, A.; Kuroda, S.;
412
Tokuyama, S.; Kikkawa, U.; Yamane, T.; Fukuda, H.. Purification,
413
characterization, and sequence determination of phospholipase D
414
secreted by Streptoverticillium cinnamoneum. J. Biochem. 1999, 125,
415
263–269.
416
(10)
Zhang, Y.-N.; Lu, F.-P.; Chen, G.-Q.; Li, Y.; Wang, J.-L. Expression,
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
417
purification, and characterization of phosphatidylserine synthase from
418
Escherichia coli K12 in Bacillus subtilis. J. Agric. Food Chem. 2009, 57,
419
122–126.
420
(11) Hosokawa, M.; Shimatani, T.; Kanada, T.; Inoue, Y.; Takahashi, K.
421
Conversion to docosahexaenoic acid-containing phosphatidylserine from
422
squid skin lecithin by phospholipase D-mediated transphosphatidylation.
423
J. Agric. Food Chem. 2000, 48, 4550–4554.
424
(12) Bi, Y. H.; Duan, Z. Q.; Li, X. Q.; Wang, Z. Y.; Zhao, X. R. Introducing
425
biobased ionic liquids as the nonaqueous media for enzymatic synthesis
426
of phosphatidylserine. J. Agric. Food Chem. 2015, 63, 1558–1561.
427
(13) Duan, Z.-Q.; Hu, F. Highly efficient synthesis of phosphatidylserine in the
428
eco-friendly solvent γ-valerolactone. Green Chem. 2012, 14, 1581.
429
(14) Dittrich, N.; Ulbrich-Hofmann, R. Transphosphatidylation by immobilized
430
phospholipase D in aqueous media. Biotechnol. Appl. Biochem. 2001,
431
34 , 189–194.
432
(15) Iwasaki, Y.; Mizumoto, Y.; Okada, T.; Yamamoto, T.; Tsutsumi, K.;
433
Yamane, T. An aqueous suspension system for phospholipase D-
434
mediated synthesis of PS without toxic organic solvent. J. Am. Oil Chem.
435
Soc. 2003, 80, 653–657.
436
(16) Pinsolle, A.; Roy, P.; Buré, C.; Thienpont, A.; Cansell, M. Enzymatic
437
synthesis of phosphatidylserine using bile salt mixed micelles. Colloids
438
Surf. B. Biointerfaces 2013, 106, 191–197.
ACS Paragon Plus Environment
Page 20 of 30
Page 21 of 30
Journal of Agricultural and Food Chemistry
439
(17) European pharmacopoeia, 8th ed.; 2013.
440
(18) Establishing, P. F. O. R.; Limits, E.; For, O.; Levels, D.; Of, C.; Solvents,
441
R.; Assessment, B. Y. R. U.S. Pharmacopeia / National Formulary; The
442
United States Pharmacopeial Convention, 2007.
443
(19) Code of Federal Regulations; 2015; Vol. 3.
444
(20) Hernandez, O. SIDS Initial Assessment Report ( SIAR ) for the 9th SIAM.
445
1999, 1–118.
446
(21) Leiros, I.; Mcsweeney, Sh. E. The reaction mechanism of phospholipase
447
D from Streptomyces sp. strain PMF. Snapshots along the reaction
448
pathway reveal a pentacoordinate reaction intermediate and an
449
unexpected final product. J. Mol. Biol. 2004, 339, 805–820.
450
(22) Bucatariu, F.; Ghiorghita, C. A.; Simon, F.; Bellmann, C.; Dragan, E. S.
451
Poly(ethyleneimine) cross-linked multilayers deposited onto solid
452
surfaces and enzyme immobilization as a function of the film properties.
453
Appl. Surf. Sci. 2013, 280, 812–819.
454
(23) Gun’ko, V. M.; Mikhailova, I. V.; Zarko, V. I.; Gerashchenko, I. I.; Guzenko,
455
N. V.; Janusz, W.; Leboda, R.; Chibowski, S. Study of interaction of
456
proteins with fumed silica in aqueous suspensions by adsorption and
457
photon correlation spectroscopy methods. J. Colloid Interface Sci. 2003,
458
260, 56–69.
459
(24) Voronin, E. F.; Guzenko, N. V; Pakhlov, E. M.; Nosach, L. V; Leboda, R.;
460
Malysheva, M. L.; Borysenko, M. V; Chuiko, A. A. Interaction of poly
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
461
( ethylene oxide ) with fumed silica. 2004, 279, 326–340.
462
(25) Gun’ko; Gun’ko, V. M.; Voronin; Voronin, E. F.; Nosach; Nosach, L. V;
463
Pakhlov; Pakhlov, E. M.; Guzenko; Guzenko, N. V; et al. Adsorption and
464
Migration of Poly(vinyl pyrrolidone) at a Fumed Silica Surface. Adsorpt.
465
Sci. Technol. 2006, 24, 143–157.
466
(26) Juneja, L. R.; Toru, K.; Tsuneo, Y.; Shoichi, S. Kinetic evaluation of
467
conversion of phosphatidylcholine to phosphatidylethanolamine by
468
phospholipase D from different sources. Biochim. Biophys. Acta
469
(BBA)/Lipids Lipid Metab. 1988, 960, 334–341.
470
(27) Savaji, K.; Li, X.; Couzis, A. Understanding the lateral movement of
471
particles adsorbed at a solid-liquid interface. J. Colloid Interface Sci. 2015,
472
453, 276–280.
473
(28) Talbert, J. N.; Goddard, J. M. Colloids and Surfaces B : Biointerfaces
474
Enzymes on material surfaces. Colloids Surfaces B Biointerfaces 2012,
475
93, 8–19.
476
(29) Jia, H.; Zhu, G.; Wang, P. Catalytic behaviors of enzymes attached to
477
nanoparticles: the effect of particle mobility. Biotechnol. Bioeng. 2003, 84,
478
406–414.
479
(30) Wu, C.-S.; Lee, C.-C.; Wu, C.-T.; Yang, Y.-S.; Ko, F.-H. Size-modulated
480
catalytic activity of enzyme-nanoparticle conjugates: a combined kinetic
481
and theoretical study. Chem. Commun. 2011, 47, 7446–7448.
482
(31) Pinsolle, A.; Roy, P.; Buré, C.; Thienpont, A.; Cansell, M. Enzymatic
ACS Paragon Plus Environment
Page 22 of 30
Page 23 of 30
Journal of Agricultural and Food Chemistry
483
synthesis of phosphatidylserine using bile salt mixed micelles. Colloids
484
Surfaces B Biointerfaces 2013, 106, 191–197.
485
(32) Damnjanović, J.; Iwasaki, Y. Phospholipase D as a catalyst: Application
486
in phospholipid synthesis, molecular structure and protein engineering. J.
487
Biosci. Bioeng. 2013, 116, 271–280.
488
(33) Deyonker, N. J.; Webster, C. E. Phosphoryl Transfers of the
489
Phospholipase D Superfamily: A Quantum Mechanical Theoretical Study.
490
J. Am. Chem. Soc. 2013, 135, 13764–13774.
491
(34) Rodrigues, R. C.; Ortiz, C.; Berenguer-Murcia, Á.; Torres, R.; Fernández-
492
Lafuente, R. Modifying enzyme activity and selectivity by immobilization.
493
Chem. Soc. Rev. 2013, 42, 6290–6307.
494
(35) Hudson, E. P.; Eppler, R. K.; Clark, D. S. Biocatalysis in semi-aqueous
495
and nearly anhydrous conditions. Curr. Opin. Biotechnol. 2005, 16 (6),
496
637–643.
497
(36) Juneja, L. R.; Taniguchi, E.; Shimizu, S.; Yamane, T. Increasing
498
productivity by removing choline in conversion of phosphatidylcholine to
499
phosphatidylserine by phospholipase D. J. Ferment. Bioeng. 1992, 73,
500
357–361.
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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.
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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
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Figure 1
Figure 2
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Figure 3
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Figure 4
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TOC Graphic
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