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Chemical amination of Rhizopus oryzae lipase for multipoint covalent immobilization on epoxy-functionalized supports: Modulation of stability and selectivity
Accepted Manuscript Title: Chemical amination of Rhizopus oryzae lipase for multipoint covalent immobilization on epoxy-functionalized supports: Modulation of stability and selectivity Author: Maryam Ashjari Mehdi Mohammadi Rashid Badri PII: DOI: Reference:
S1381-1177(15)00052-1 http://dx.doi.org/doi:10.1016/j.molcatb.2015.02.011 MOLCAB 3115
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
29-12-2014 17-2-2015 18-2-2015
Please cite this article as:http://dx.doi.org/10.1016/j.molcatb.2015.02.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chemical amination of Rhizopus oryzae lipase for multipoint covalent immobilization on epoxy-functionalized supports: modulation of stability and selectivity
5
Maryam Ashjaria,b, Mehdi Mohammadi*c, Rashid Badria,b
ip t
1 2 3 4
6 7
a
8
Iran.
9
b
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Department of Chemistry, College of Science, Ahvaz Branch, Islamic Azad University, Ahvaz,
Department of Chemistry, Khouzestan Science and Research Branch, Islamic Azad University,
Ahvaz, Iran.
11
c
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National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O.Box:14965/161
13
Tehran, Iran.
an
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M
Bioprocess Engineering Department, Institute of Industrial and Environmental Biotechnology,
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te
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Ac ce p
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*
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Phone: (+98) 21 44580461
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Fax: (+98) 21 44580399
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E-mail: [email protected]
Corresponding author
23 24 25 1 Page 1 of 29
Abstract
27
Multipoint covalent attachment of Rhizopus oryzae lipase (ROL) on epoxy-functionalized silica
28
and silica nanoparticles (MCM-41 and SBA-15) is reported. Multipoint immobilization of
29
enzymes on these supports usually performs by the reaction between several epoxy groups of the
30
support and several Lys residues on the external surface of the enzyme molecules at pH 10.
31
However, this standard immobilization procedure is unsuitable for ROL due to the low stability
32
of ROL at pH 10. Introducing new amino groups with lower pKb to the surface of ROL by using
33
chemical amination strategy permits immobilization of the enzyme at lower pH values.
34
Immobilization/stabilization of aminated ROL was performed in two steps. First the enzyme is
35
covalently immobilized at pH 7.0 and then the already immobilized enzyme is further incubated
36
at pH 9.2 to promote the formation of further covalent linkages between the immobilized ROL
37
and the support. The results showed higher thermal and co-solvent stability for immobilized
38
derivatives of aminated ROL compared to the results obtained for the derivatives of not-
39
aminated ROL and free ROL. Influence of the immobilization procedure on selectivity of the
40
immobilized preparations was studied in selective hydrolysis of fish oil at three different
41
conditions. The selectivity and reusability of ROL was greatly improved after immobilization.
42
All the derivatives discriminate between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-
43
4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA.
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Keywords: Epoxy supports, Fish oil, Rhizopus oryzae lipase, Multipoint covalent
46
immobilization, Silica nanoparticles
47 2 Page 2 of 29
1. Introduction
49
Lipases represent a class of enzymes with important roles in many essential physiological
50
processes [1]. They are also involved in a wide variety of applications in detergent and food
51
industries, leather industry, environmental management, cosmetics and perfume industry,
52
biomedical applications and biosensors [2-4], The lipase from Rhizopus Oryzae is a 1,3-specific
53
and moderately stable enzyme which is commercially available in both soluble and immobilized
54
form [5] . ROL has a molecular size of 32,000 Da and a pI of 7.6. It has 21 residues of aspartic
55
(12) and glutamic (9) groups which is higher than the number of lysine moieties (15 residues).
56
Regarding the high selectivity of ROL, several applications of this enzyme in hydrolysis and
57
esterification of fish oils were reported in literature [6, 7]. The beneficial health effects of fish oil
58
are now well documented and attributed to omega-3 polyunsaturated fatty acids (n-3 PUFA), cis-
59
5,8,11,15,17-eicosapentaenoic acid and cis-4,7,10,13,16,19-docosahexaenoic acid in particular
60
[8]. Due to the importance of polyunsaturated fatty acids, various techniques have been used for
61
enrichment of these compounds. One of the most promising techniques is the use of lipase-
62
catalyzed enzymatic hydrolysis of fish oil [9]. Lipases, both in soluble and immobilized form
63
discriminate against EPA and DHA; the main bioactive fatty acids in fish oil [10].
64
The improvement of efficiency of lipases in chemical reactions is still one of the main issues for
65
their effective application as industrial biocatalysts. Immobilization of lipase on solid supports is
66
the most known methods for such improvements [11]. Several attempts have been carried out on
67
the preparation of immobilized lipases, which involves a variety of immobilization techniques
68
and new support materials [12]. Enzyme immobilization permits to obtain a heterogeneous
69
catalyst, and if properly designed, improved stability and selectivity of the enzyme can be
70
expected.
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The most of strategies for covalent immobilization of enzymes is based on using the amino
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groups of the Lys residues which are the most abundant nucleophilic groups of the protein
73
surface [13]. The most obstacle of using these groups for immobilization of enzymes is that they
74
are reactive only at pH values over 10. Introducing new amino groups with lower pH values on
75
the protein surface via chemical amination of the carboxylic groups of Asp and Glu is a well
76
described method to optimize immobilization process [14]. In this way, the lower pKb of the new
77
amino groups allows to immobilize the enzyme via multipoint covalent attachment under milder
78
condition [15]. This type of irreversible immobilization is particularly preferred when the pH
79
stability of the used enzyme is relatively low. Moreover chemical amination increases the
80
number of interactions between enzyme and activated supports, promoting a higher number of
81
covalent attachments that increase enzyme stabilization.
82
Epoxy-activated supports are usually used to perform immobilization of proteins and enzymes at
83
mild conditions. These activated supports can be stored for a long time and have high stability at
84
neutral pH values [16]. At this condition epoxy groups can react with nucleophilic groups
85
present on the protein surface; in particular with terminal amino groups. However, it is well
86
documented that much stabilization of the enzyme could be expected if the immobilization
87
process occurs through several residues [17]. Immobilization of different enzymes on epoxy
88
supports via multipoint covalent attachment has been reported as an efficient way to improve the
89
enzyme stability [18].
90
chymotrypsin
91
immobilization/stabilization procedure [19]. To promote multipoint covalent attachment they
92
incubated the already immobilized enzyme under more drastic conditions (pH> 10 and long time
93
incubation). The results showed that the stability of the final derivatives was remarkably
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on
Mateo et al. reported immobilization of penicillin G acylase and
epoxy-activated
support
(Eupergit
C)
via
a
three-step
4 Page 4 of 29
increased by favoring the additional linkage between the immobilized enzyme molecule and the
95
support. Some other reports also pointed the positive effect of multipoint covalent attachment of
96
enzymes on epoxy supports to improve functional properties of the immobilized enzymes. To the
97
best of our knowledge, there is no report on immobilization of ROL on epoxy-functionalized
98
siliceous materials. The strategy of chemical amination to perform a stabilized–immobilized
99
derivative of ROL on epoxy-functionalized supports is also reported for the first time. To check
100
the effect of chemical amination-immobilization of ROL on its selectivity, we have used the
101
selective hydrolysis of fish oil in a biphasic system.
102
2. Materials and methods
103
2.1 Materials
104
The lipase from Rhizopus oryzae, fish oil from menhaden (containing 10-15% of
105
eicosapentaenoic
106
docosahexaenoic acid, ethylenediaminetetraacetic acid (EDTA), p-nitrophenyl butyrate (p-NPB),
107
sodium silicate, tetra ethyl ortho silicate (TEOS), polyuronic acid (P123), sodium periodate,
108
ethylenediamine
109
glycidoxypropyltrimethoxylsilane (GPTMS) were from Sigma. 1,4-dioxane, 1-propanol, 2-
110
propanol and silica gel (70 -230 mesh) were purchased from Merck. cis-5,8,11,14,17-
111
Eicosapentaenoic acid was from Cayman company. Other reagents and solvents were of
112
analytical or HPLC grade. Fourier transform infrared spectra (FT-IR) were recorded on a Bomen
113
FT-IR-MB-series instrument with a KBr pellet technique. Thermogravimetry (TGA) and
114
differential thermal analysis (DTA) were carried out from 10°C to 800 °C at a heating rate of 20
115
°C/min in air atmosphere using a STA 503M system from Bahr GmbH, Germany.
116
2.2. Methods
an
8-15%
M
and
of
docosahexaenoic
acid),
cis-4,7,10,13,16,19-
1-ethyl-3-(dimethylaminopropyl)
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(EDA),
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carbodiimide
(EDC)
and
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2.2.1 Preparation of the silica nanoparticles
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2.2.1.1 Preparation of SBA-15
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Pure siliceous SBA-15 was prepared using a previously reported procedure [20]. Pluronic P123
120
triblock copolymer (EO20-PO70-EO20, BASF) was used as template. Briefly, four grams of
121
Pluronic P123 were added to 144 mL of an aqueous solution of HCl (2 M) at 40 °C.
122
Successively, TEOS was added dropwise (mass ratio TEOS/P123= 2:1) and then stirred for 2 h.
123
Afterwards, the mixture was transferred to Teflon-lined sealed container and kept at 100 °C for
124
48 h. The white solid was filtered, washed with distilled water and calcined at 550°C to remove
125
the template.
126
2.2.1.2 Preparation of MCM-41
127
Sodium silicate (19.0 g, 27% SiO2, diluted with 40.6 mL of distilled water) was mixed with a
128
solution of 16.4 g cetyltrimethylammonium bromide (CTAB) in 69.2 mL of distilled water. The
129
sodium silicate/CTAB solution was adjusted to pH 11.0 and stirred for 30 min. Finally the
130
mixture transferred into a stainless steel jacketed Teflon vessel and heated at 100 °C for 48 h.
131
Calcination of the obtained solid was carried out at 550 °C to remove the template [21].
132
2.2.2. Functionalization of silica, SBA-15 and MCM-41,
133
Surface modification of silica, SBA-15 and MCM-41 was performed according to the
134
literature[22]. Briefly, the dry mesoporous silica materials (1 g) were dispersed in 50 mL of dry
135
toluene; then 1-2 mL of GPTMS and 0.15 mL Et3N were added. The resulting mixture was
136
refluxed under nitrogen atmosphere and vigorous stirring for 4 h. The modified nanoparticles
137
were collected by filtration and washed thoroughly with THF. Finally the modified particles
138
were dried at 120 °C for 8 h.
139
2.2.2.1 Determination of epoxy groups on the support
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Determination of the epoxy groups on the support was carried out as follows: 200 mg of the
141
support was added to 1.5 mL of 1.3 M sodium thiosulphate solution and then the solution was
142
titrated by addition of 0.1 M hydrochloric acid until neutralization. The amount of epoxy groups
143
was calculated from the amount of hydrochloric acid needed to maintain neutrality of the
144
mixture. The same reaction was performed using unmodified nanoparticles as blank [23].
145
2.2.3. Chemical amination of ROL.
146
Chemical modification of the carboxyl groups of ROL was performed according to the method
147
described by Hoare and Koshland [24]. Briefly the amount of 300 µL of an aqueous solution of
148
ethylenediamine (1 M) was added to 10 mL of a solution containing ROL (1 mg/mL) in distilled
149
water. Subsequently to initialize the reaction 10 mM of 1-ethyl-3-(dimethylaminopropyl)
150
carbodiimide was added and the pH was adjusted to 4.7. After 3h the reaction mixture was
151
extensively dialyzed against water.
152
2.2.4. Enzymatic activity assay
153
The activities of the soluble lipase and its immobilized preparations were analyzed
154
spectrophotometrically by measuring the increment in absorbance at 348 nm (= 5150 M-1cm-1).
155
The increase in absorbance produced by the release of p-nitrophenol in the hydrolysis of p-NPB
156
in 25 mM sodium phosphate buffer at pH 7.0 and 25°C. Briefly, 0.01-0.1 mL of the lipase
157
suspension or solution (blank or supernatant without further dilution) was added to 1.25 mL of
158
substrate solution (0.8 mM) under magnetic stirring [15]. Spontaneous hydrolysis of p-NPB was
159
measured using 1.25 mL of substrate solution (0.8 mM) in the absence of ROL as control.
160
Enzymatic activity is given as 1µmol of p-nitrophenol released per minute per mg of the enzyme
161
(IU) under the condition described above.
162
2.2.5 Enzyme immobilization on epoxy-functionalized supports
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2.2.5.1 Immobilization of not-aminated ROL on epoxy-functionalized supports
164
50 mg of epoxy-functionalized SBA-15, MCM-41 and 300 mg of epoxy-functionalized silica
165
was incubated in 3 mL of 25 mM potassium phosphate buffer at pH 7.0 containing (1 mg/mL) of
166
ROL at 25 °C for 24 h. Periodically, samples of the supernatants were withdrawn and analyzed
167
for determination of the protein concentration by the Bradford's method [25]. Finally the
168
immobilized ROL derivatives were filtered and washed by distilled water and stored at 4°C.
169
2.2.5.2 Immobilization of aminated ROL on epoxy-functionalized supports
170
To perform the first immobilization step, 3 mL of aminated ROL (1 mg/mL) was offered to 50
171
mg of epoxy-functionalized SBA-15, MCM-41 and 300 mg of epoxy-functionalized silica at pH
172
7.0 at 25 °C. After immobilization of the soluble aminated enzyme (disappearance of hydrolytic
173
activity of the supernatant) the final pH of the solution was adjusted to 9.2 and the suspension
174
was incubated over night at 4°C under mild stirring. Finally the suspension was filtered and
175
washed with abundant distilled water.
176
2.2.5.3. Determination of the amount of protein bonded to the carriers
177
The amount of protein in supernatant and blank was determined by the Bradford method. The
178
amount of immobilized ROL was calculated by subtracting the amount of the enzyme in
179
supernatant from the total amount of the lipase used for immobilization. The reported yields of
180
immobilization were calculated as the ratio of the amount of the protein on the support to the
181
initial amount. Yields were expressed as percentage.
182
2.2.5.4. Leaching experiment
183
100 mg of each biocatalyst was incubated in a solution containing 1 M of NaCl with vigorous
184
magnetic stirring for 24 h. Then the concentration of ROL in the supernatant was measured by
185
the Bradford's method.
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2.2.6. Thermal inactivation of different ROL immobilized preparations
187
Free enzyme and immobilized preparations of ROL were incubated in 25 mM sodium phosphate
188
at pH 7.0 and different temperatures. The suspension of each sample was withdrawn periodically
189
and their activities were measured using the p-NPB assay.
190
2.2.7. Co-solvent stability of ROL and immobilized preparations
191
Free enzyme and immobilized preparations of ROL were incubated in a total volume of 1 mL
192
solution containing 25 mM sodium phosphate buffer pH 7.0 and 10% and 20% of 1,4-dioxane, 1-
193
propanol and isopropanol at 25°C. The suspension of each sample was periodically withdrawn
194
and their activities checked with the enzymatic activity assay as described above.
195
2.2.8. Hydrolysis of Fish Oil
196
Hydrolysis of fish oil was carried out in a biphasic system containing aqueous and organic
197
solvent [26]. 4.5 mL of cyclohexane, 500 µL of fish oil and 5mL of phosphate buffer (25 mM)
198
pH 5.0 and 7.0 were added in a test tube and pre-incubated at 25°C for 15 min with vigorous
199
stirring. To start the reaction, 50 mg of immobilized preparations or a solution containing 3 mg
200
of the free lipase were added to the reaction medium. Progress of the reaction was followed by
201
taking 100 µL of organic phase at selected time intervals followed by addition of 200 µL of 2-
202
propanol. Afterward, the selectivity and hydrolytic activity of each derivative were evaluated by
203
using the reverse-phase HPLC (Knauer with an UV detector) on a Grace C4 (25cm ×0.46cm).
204
The mobile phase was 55% of acetonitrile/45% of 10 mM ammonium phosphate (V/V) at pH 3.0
205
at flow rate of 0.4 mL/min and 210 nm in the UV detector. The retention times for the
206
unsaturated fatty acids were 25 and 29 min for EPA and DHA respectively. These enzymatically
207
produced PUFA were compared to their corresponding pure commercial standards.
208
2.2.9. Recyclability of immobilized derivatives
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The recyclability was studied by determining activity of immobilized derivatives in subsequent
210
reactions relative to that of the first reaction (pH 7.0 and 25°C). After each cycle (8h), enzyme
211
loaded particles were washed with cyclohexane and re-introduced into a fresh reaction medium
212
for another assay run and this procedure was repeated up to five cycles in the same condition.
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3. Result and Discussion 3.1. Preparation, functionalization and quantification of the supports
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Siliceous materials have become a common choice of supports for enzyme immobilization.
219
Insolubility in water, high enzyme loading capacity, mechanical strength, high reactivity towards
220
functionalizing agents are the unique advantages of these materials. Their porous structure
221
creates a protective environment where the enzymes can tolerate more extreme pH, elevated
222
temperature and higher salt concentrations. Commercially available silica gel with moderate
223
surface areas (500 m2/g) and mesopuros silica nanoparticles (SBA-15, MCM-41) with ordered
224
pore structure were used for immobilization of ROL. Large-pore SBA-15 with surface area of
225
952 m2/g and pore sizes of 10.2 nm and MCM-41 with surface area of 1275 m2/g and pore sizes
226
of 3.9 nm were synthesized according to our previously published paper [27]. The epoxy-
227
functionalization
228
glycidoxypropyltrimethoxylsilane (Scheme 1). Quantification of oxirane groups on these
229
supports was performed by titration of the released hydroxide ion from the reaction between
230
epoxy groups on the support and sodium thiosulphate. The results revealed that the amount of
231
epoxy groups on the surface of silica, SBA-15 and MCM-41 was about 277, 805 and 850 µmol
232
per gram of each support, respectively. High degree of functionalization of SBA-15 and MCM-
233
41 compared to silica can be attributed to more surface area of these supports.
234
3.2. Multipoint covalent immobilization of ROL
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of
silica,
SBA-15
and
MCM-41
was
carried
out
using
3-
10 Page 10 of 29
Multipoint covalent attachment of enzymes on activated supports is a very powerful tool for
236
stabilizing proteins. The stabilization factor of the immobilized enzyme depends on the number
237
of linkages between support and enzyme. The formation of multiple covalent bonds, keeps the
238
relative positions of all the groups involved in immobilization unchanged during conformational
239
change induced by any distorting agent (heat, organic solvents, extreme pH values). This strategy
240
is usually used for immobilization of enzymes on aldehyde-functionalized supports [28].
241
However there are few reports of multipoint attachment of enzymes on epoxy-functionalized
242
supports [17-19]. These investigations are almost based on incubation of enzymes at high pH
243
values (pH>10) to promote several linkages between amino groups of Lys residues and the
244
epoxy groups of the support. Our investigation showed that ROL is clearly unstable at pH 10;
245
making impossible its multipoint covalent attachment on the epoxy-functionalized supports via
246
Lys residues. Therefore new amino groups with lower pkb were introduced to the surface of the
247
enzyme by using the chemical amination strategy. In this way ethylenediamine was used to
248
amination of Asp and Glu residues. The lower pkb of the new amino groups permits
249
immobilization of ROL at lower pH values, where the enzyme is completely stable. The
250
chemical amination of the protein surface was performed via the reaction between
251
ethylenediamine and carboxylic groups of the free lipase, after activation with carbodiimide. The
252
results showed that chemical amination has low impact on the enzyme activity; decreasing only
253
5-8% of its initial activity. The loss of 3-33% in enzyme activity after chemical amination of
254
penicillin G acylase and glutaryl acylase at different conditions have previously reported [29].
255
The aminated enzyme was immobilized on the supports via a two-step procedure. First most of
256
the enzyme was covalently immobilized under very mild experimental conditions (pH 7.0 and
257
25°C). The most reactive group at this condition is the terminal amino groups of the protein.
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Then the already immobilized enzyme was further incubated at pH 9.2 to facilitate the formation
259
of new covalent linkages between the immobilized enzyme molecule and the support. In this
260
way, all stabilizing advantages already achieved by the first immobilization process will be taken
261
to facilitate the long-term incubation of the enzyme derivative under hard experimental
262
condition. The immobilization of not-aminated ROL on each support was also performed at pH
263
7.0 and 25°C. As can be seen from table 1, almost complete immobilization of aminated and not-
264
aminated ROL on silica-epoxy achieved; producing 7.2 and 6.5 U/mg specific activity,
265
respectively. Compared to the specific activity of the soluble enzyme (7.7 U/mg enzyme), it
266
shows about 6-14% reduced activity. In order to perform immobilization on functionalized SBA-
267
15 and MCM-41, a solution containing 60 mg of ROL was offered to 1g of each support at low
268
ionic strength (25 mM). As can be seen in table 1, 85 and 86% of aminated and not-aminated
269
ROL is bound to SBA-epoxy after 24h of incubation respectively. The specific activity of
270
aminated ROL showed about 30% decrease after immobilization on this support. It seems that
271
the porous structure of SBA-15 and higher amount of immobilized enzyme compared to silica-
272
epoxy, increases diffusion limitation of substrate/product. Low immobilization yield (63-65 %)
273
was obtained in immobilization of aminated/not-aminated ROL on MCM-epoxy support. It is
274
most likely because of the fact that the small pore size (3.9 nm) of this support is not adequate to
275
make the internal surface accessible for immobilization of the enzyme. In other worlds, the
276
enzyme gets stuck in the pore entrances subsequently blocking the pore. The specific activity of
277
both aminated and not-aminated ROL decreased after immobilization on MCM-epoxy.
278
Generally there are many reports of decrease in enzyme activity after covalent immobilization on
279
different supports [30]. It can be resulted by some phenomena like denaturation of the protein
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during immobilization, altering microenvironment of the enzyme and diffusion limitation after
281
immobilization of enzyme.
282
In order to ensure the covalent attachment of ROL on the modified supports; leaching
283
experiments were performed. The condition of desorption of the protein from the supports was
284
examined by incubation of all the immobilized derivatives in a solution containing 1M NaCl at
285
25 °C for 24h. The activity of the supernatant was measured by p-NPB hydrolysis without
286
showing any measurable activity. The Bradford assay showed also no detectable enzyme in
287
solution clearly proving that the immobilization is exclusively performed via covalent binding.
288
3.3. Thermal stability of free and immobilized derivatives of ROL
289
Increased stability is one of the most common expected results of covalent immobilization of
290
enzymes and is often an important economic factor. Thermal stability of free ROL and the
291
immobilized derivatives was investigated at different temperatures. As figure 1 shows the
292
soluble enzyme is relatively unstable and loses 64, 82, 96% of its activity after 2h incubation at
293
45, 50 and 55 °C respectively. Rapid inactivation of the soluble ROL reveals the necessity of
294
using immobilization techniques to improve its stability. The results showed that immobilization
295
of non-modified ROL on silica-epoxy improved its thermal stability; remaining about 50% of its
296
initial activity after 2h incubation at 50 °C. Improved stability of ROL immobilized on MCM-
297
epoxy and SBA-epoxy compared to the free enzyme is also observed. These derivatives are
298
completely stable after 2h incubation at 45 °C. Figure 1 clearly shows the positive effect of
299
multipoint covalent attachment of enzyme on epoxy-functionalized supports. All the derivatives
300
are quite stable at 45 °C; keeping 100% of their initial activities after 2h of incubation. While the
301
free enzyme is almost inactive at 55 °C, silica-NH2-ROL, SBA-NH2-ROL and MCM-NH2-ROL
302
keeps 38%, 60% and 57% of their initial activities at the same condition, respectively. These
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higher stabilities of aminated ROL immobilized on epoxy supports are due to possibility of
304
multipoint covalent attachment with the epoxy groups, and the fact that in the used condition (pH
305
9.2) not only terminal amino groups may react with the supports, but also chemically introduced
306
amino groups.
307
3.4. Solvent stability of free and immobilized derivatives of ROL
308
The use of organic solvents for enzymatic reactions has demonstrated to be a useful method to
309
increase the efficiency of biocatalysts [31]. Most of the enzymes are usually not suited for
310
application in non-aqueous media in industrial processes. It has been reported that the polar
311
solvents can interact with the enzyme and reduce its catalytic activity [31]. This is due to the fact
312
that a few amounts of water molecules are required for enzymatic function. Organic solvents,
313
particularly those having log P values below 2 strongly distort this essential water-enzyme
314
interaction thereby inactivating the enzyme [32]. The use of immobilized form of enzymes is
315
considered as an efficient way to improve their stabilities in presence of organic solvents [33, 34]
316
The effect of immobilization on co-solvent stability of ROL was investigated in presence of three
317
water-miscible solvents (10% and 20% of 1-propanol, 2-propanol and dioxane) (Figure 2). These
318
polar organic solvents with log P<2 make a harsh condition to evaluate the stability of the
319
derivatives. The result showed that the soluble enzyme retains 72-91% of its initial activity after
320
24h of incubation in presence of 10% of each solvent. However, after increasing percentage of
321
the solvents to 20%, remarkable decrease in enzyme activity is observed especially for 1-
322
propanol. As figure 2 shows immobilization of ROL on the supports clearly improves its co-
323
solvent stability. SBA-ROL and MCM-ROL show slightly higher stabilities in presence of 10
324
and 20% of the solvents compared to silica-ROL. This might be explained by altering
325
microenvironment of the immobilized enzyme due to different nature of the micro and nano
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14 Page 14 of 29
structure of the supports. However, limitations in substrate access, immobilization of the enzyme
327
from different side and unfavourable enzyme conformation may also be contributing factors. The
328
most promising results obtained from the investigation of the stability of aminated ROL
329
immobilized on different supports. As can be concluded from figure 2, chemical amination
330
greatly improves the stability of immobilized derivatives. SBA-NH2-ROL retains its whole
331
activity after 24h incubation in presence of 10 and 20% of each solvent. These results suggest
332
high ability of chemical amination approach to produce derivatives suitable to use in chemical
333
reactions in which presence of organic solvents and long time of reaction are needed.
334
3.5. Fish oil hydrolysis
335
It has been reported that chemical amination and subsequent immobilization of the aminated
336
enzymes leads to modulate their catalytic properties (selectivity and activity) [14]. Alterations of
337
the shape and size of the active centre has been proposed as a possible cause of such modulation.
338
By using free and immobilized derivatives of ROL the hydrolysis of fish oil from menhaden in a
339
biphasic system containing aqueous and organic solvent was studied. HPLC-UV analysis was
340
used to follow the rate of PUFAs release and the selectivity of the preparations against EPA and
341
DHA as the most valuable ingredients of the oil. The Selectivity is calculated as the ratio
342
between released EPA and released DHA and activity was calculated by the following equation:
an
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Activity =
343
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326
poly unsaturated fatty acid concentration (mmol) enzyme (mg) × time (minute)
344
As same as the results obtained in p-NPB reaction (table 1) covalent immobilization of ROL
345
causes to decrease in enzyme activity. The derivatives obtained from immobilization of
346
chemically aminated enzyme produce lower activities compared to the not-aminated
347
preparations. Between the three selected conditions for the hydrolysis reaction, pH 7.0 and 25 °C
348
were the optimal condition in terms of enzymatic activity. Beside of activity, the EPA/DHA 15 Page 15 of 29
selectivity of the used biocatalyst is also a critical parameter in fish oil hydrolysis. As EPA and
350
DHA are very similar and difficult to be separated by using physico-chemical protocols,
351
selective hydrolysis of fish oil can be a powerful to produce almost pure EPA. As can be seen in
352
table 2, while the free enzyme poorly discriminates between EPA and DHA, its selectivity
353
greatly improves after immobilization both in aminated and not-aminated forms. Wide range of
354
selectivities (3.1-13.5) were produced depends on the condition of reaction and the kind of
355
procedure/support used for immobilization. All the derivatives were also observed to display a
356
significant preference for EPA as compared to DHA which is in accordance with previous
357
reports [10, 26]. As the results show, lowering the temperature causes to significant improvement
358
in enzyme selectivity. Moreover the derivatives of chemically aminated ROL shows higher
359
selectivity compared to not-aminated ROL preparations. While the selectivity of the soluble
360
ROL is 2.8 at pH 5.0 and 4°C, SBA-NH2-ROL shows almost 5 fold improvement in selectivity at
361
the same condition. The observed selectivity of this derivative permits the production of PUFA
362
with almost 93 % of EPA purity at the first stages of the reaction. In a previously published
363
report Fernandez et al. have investigated on EPA/DHA selectivity of 7 different lipases
364
immobilized on octyl-sepharose and cyanogen bromide-sepharose. The obtained selectivities
365
(6.9 and 9.8 for octyl-ROL and CNBr-ROL, respectively) were the best results of the examined
366
biocatalysts [7].
367
Insignificant improvement in selectivity was observed for the derivatives obtained from
368
immobilization of both aminated and not-aminated ROL on epoxy-functionalized MCM-
369
41(MCM-ROL and MCM-NH2-ROL). This observation clearly demonstrates that apart from the
370
reaction condition and modification of the enzyme surface, the size and shape of the support can
371
influence the catalytic properties of the enzyme. Regarding the activity and selectivity of the
Ac ce p
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16 Page 16 of 29
derivatives, the best results were obtained for silica-ROL with 13.5 selectivity and 0.02 (U/mg
373
lipase per minute) at pH 5.0 and 4 °C.
374
3.3.5. Recyclability of immobilized derivatives
375
One of the technological and economical advantages of enzyme immobilization is the ability of
376
the immobilized preparations to repeatedly use in chemical reactions. In order to investigate
377
reusability of the immobilized derivatives they were used in hydrolysis reaction for five cycles.
378
After each run (8 h), the immobilized preparations were recovered by filtration, washed with
379
cyclohexane and reused for a new reaction under the same conditions. The activity of first cycle
380
of the reaction was set as 100% and the activity in the subsequent reactions was calculated
381
accordingly. As figure 3 shows the derivatives retain about 81-94% of their activities after five
382
cycles of the reaction. The results also present a meaningful improvement of recyclability of the
383
three derivatives of aminated ROL confirming that chemical amination has positive effect on
384
enzymatic function of ROL. The best result of reusability was obtained for SBA-NH2-ROL with
385
97% retaining of its activity after five cycles of the reaction.
cr
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386
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372
387
4. Conclusion
388
Covalent immobilization of enzymes on epoxy-functionalized supports is normally carried out
389
under very mild conditions. Under these conditions the intense multipoint covalent linkage
390
between the immobilized enzyme and the support is not possible. This is due to the low
391
reactivity of the epoxy groups and the reactive groups of the protein at pH 7. Therefore
392
producing an effective strategy to broaden thermal and co-solvent stability and selectivity of
393
ROL was considerable target of this research. For this purpose, chemical amination of ROL and
394
immobilization of aminated/not-aminated ROL on different epoxy-functionalized supports were
17 Page 17 of 29
performed. Chemical amination introduces new amino groups with a lower pKb value than that
396
of the Lys residues. The derivatives of the chemically aminated ROL are more stable than
397
derivatives of the not-aminated ROL with respect to thermal and solvent inactivation. The most
398
interesting results were found for SBA-NH2 –ROL, which showed retaining of 100% of its initial
399
activity after 24h incubation in presence of 20% of 1-propanol, 2-propanol and dioxane. This
400
derivative also showed higher thermal stability compared to other derivatives. Beside of these
401
interesting results, the selectivity of the enzyme was also modulated using chemical amination
402
procedure. Although the selectivity of the derivatives from MCM-epoxy is relatively low, most
403
of the derivatives showed high selectivity in fish oil hydrolysis compared to free enzyme. The
404
results demonstrate that, for the development of an optimal catalyst for the production of omega-
405
3 fatty acids, it is necessary to consider factors such as the immobilization protocol and the kind
406
of support. Also remarkable improvement in selectivity and stability of the immobilized
407
derivatives compensates undesirable decrement of activity during chemical amination and
408
covalent immobilization of ROL.
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410
Acknowledgment
411
This work was financially supported by the Iran National Science Foundation (INSF) (grant
412
number 91004274) for which the authors are thankful.
413 414 415 416 417 418
18 Page 18 of 29
419
References
420 [1] D. Sharma, B. Sharma, A. Shukla, Biotechnology, 10 (2011) 23-40. [2] A. Pandey, S. Benjamin, C.R. Soccol, P. Nigam, N. Krieger, V.T. Soccol, Biotechnology and applied biochemistry, 29 (1999) 119-131. [3] R. Aravindan, P. Anbumathi, T. Viruthagiri, Indian Journal of Biotechnology, 6 (2007) 141158. [4] M. Yousefi, M. Mohammadi, Z. Habibi, Journal of Molecular Catalysis B: Enzymatic, 104 (2014) 87-94. [5] M. Ueda, S. Takahashi, M. Washida, S. Shiraga, A. Tanaka, Journal of Molecular Catalysis B: Enzymatic, 17 (2002) 113-124. [6] K. Bhandari, S. Chaurasia, A. Dalai, A. Gupta, K. Singh, Journal of Molecular Catalysis B: Enzymatic, 94 (2013) 104-110. [7] G. Fernández-Lorente, L. Betancor, A.V. Carrascosa, J.M. Guisán, Journal of the American Oil Chemists' Society, 88 (2011) 1173-1178. [8] E.A. de Deckere, Health aspects of fish and n-3 polyunsaturated fatty acids from plant and marine origin, in: Nutritional Health, Springer, 2001, pp. 195-206. [9] G. Fernandez-Lorente, M. Filice, D. Lopez-Vela, C. Pizarro, L. Wilson, L. Betancor, Y. Avila, J.M. Guisan, Journal of the American Oil Chemists' Society, 88 (2011) 801-807. [10] G. Fernández-Lorente, L. Betancor, A.V. Carrascosa, J.M. Palomo, J.M. Guisan, Journal of the American Oil Chemists' Society, 89 (2012) 97-102. [11] P. Adlercreutz, Chemical Society Reviews, 42 (2013) 6406-6436. [12] R.C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R. Fernández-Lafuente, Chemical Society Reviews, 42 (2013) 6290-6307. [13] A.G. Cunha, G. Fernández-Lorente, J.V. Bevilaqua, J. Destain, L.M. Paiva, D.M. Freire, R. Fernández-Lafuente, J.M. Guisán, Applied biochemistry and biotechnology, 146 (2008) 49-56. [14] R.C. Rodrigues, O. Barbosa, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R. FernandezLafuente, RSC Advances, 4 (2014) 38350-38374. [15] Z. Habibi, M. Mohammadi, M. Yousefi, Process Biochemistry, 48 (2013) 669-676.
448
[16] C. Mateo, O. Abian, G. Fernández‐Lorente, J. Pedroche, R. Fernández‐Lafuente, J.M.
449
Guisan, Biotechnology Progress, 18 (2002) 629-634.
450
[17] V. Grazú, O. Abian, C. Mateo, F. Batista‐Viera, R. Fernández‐Lafuente, J.M. Guisán,
451 452 453 454 455
Biotechnology and bioengineering, 90 (2005) 597-605. [18] F. López-Gallego, L. Betancor, A. Hidalgo, C. Mateo, J.M. Guisán, R. Fernández-Lafuente, Journal of biotechnology, 111 (2004) 219-227. [19] C. Mateo, O. Abian, R. Fernandez–Lafuente, J.M. Guisan, Enzyme and Microbial Technology, 26 (2000) 509-515.
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us
cr
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421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447
19 Page 19 of 29
ip t
cr
us
an
M
d
te
483
[20] A. Salis, M. Pisano, M. Monduzzi, V. Solinas, E. Sanjust, Journal of Molecular Catalysis B: Enzymatic, 58 (2009) 175-180. [21] K.A. Northcott, K. Miyakawa, S. Oshima, Y. Komatsu, J.M. Perera, G.W. Stevens, Chemical Engineering Journal, 157 (2010) 25-28. [22] J. Lin, J.A. Siddiqui, R.M. Ottenbrite, Polymers for Advanced Technologies, 12 (2001) 285292. [23] L. Sundberg, J. Porath, Journal of Chromatography A, 90 (1974) 87-98. [24] D.t. Hoare, D. Koshland, Journal of Biological Chemistry, 242 (1967) 2447-2453. [25] M.M. Bradford, Analytical biochemistry, 72 (1976) 248-254. [26] M. Mohammadi, Z. Habibi, S. Dezvarei, M. Yousefi, Food and Bioproducts Processing, (2014). [27] M. Mohammadi, Z. Habibi, S. Dezvarei, M. Yousefi, S. Samadi, M. Ashjari, Process Biochemistry, 49 (2014) 1314-1323. [28] R.C. Rodrigues, C.A. Godoy, G. Volpato, M.A. Ayub, R. Fernandez-Lafuente, J.M. Guisan, Process Biochemistry, 44 (2009) 963-968. [29] F. López-Gallego, T. Montes, M. Fuentes, N. Alonso, V. Grazu, L. Betancor, J.M. Guisán, R. Fernández-Lafuente, Journal of biotechnology, 116 (2005) 1-10. [30] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente, Enzyme and Microbial Technology, 40 (2007) 1451-1463. [31] F.H. Arnold, Trends in biotechnology, 8 (1990) 244-249. [32] A.M. Klibanov, Nature, 409 (2001) 241-246. [33] R. Fernandez-Lafuente, C. Rosell, L. Caanan-Haden, L. Rodes, J. Guisan, Enzyme and Microbial Technology, 24 (1999) 96-103. [34] V. Stepankova, S. Bidmanova, T. Koudelakova, Z. Prokop, R. Chaloupkova, J. Damborsky, ACS Catalysis, 3 (2013) 2823-2836.
Ac ce p
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20 Page 20 of 29
Figure captions:
484
Scheme 1. A description of the supports treatment used for immobilization.
485
Figure 1. Thermal stability of free ROL and immobilized preparations at 45°C, 50 °C, 55 °C.
486
Experimental condition: Increasing certain amount of each biocatalyst in 1mL of sodium
487
phosphate buffer 25 mM (pH 7.0) and incubation at different temperatures for 2h. Initial activity
488
of ROL and each immobilized derivatives was determined in 1 mL of sodium phosphate buffer
489
25 mM (pH 7.0) at 25 °C and set as 100%.
490
Figure 2. Co-solvent stability of free ROL and immobilized preparations in presence of 20 % of
491
organic solvents. Experimental condition: Incubation of each biocatalyst in 1 mL solution
492
containing 25 mM sodium phosphate buffer (pH 7.0) and 20% of three organic solvents at 25°C.
493
Initial activity of ROL and each immobilized derivatives was determined in 1 mL of sodium
494
phosphate buffer 25 mM (pH 7.0) at 25 °C and set as 100%.
495
Figure 3. Effect of repeated use of immobilized preparations on their activity in fish oil
496
hydrolysis. Reaction conditions: A biphasic system containing 4.5 mL of cyclohexane, 5mL (25
497
mM) of phosphate buffer (pH 7, 25°C), 500 µL of fish oil and 100 mg of biocatalyst.
cr
us
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M
Ac ce p
te
d
498 499
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483
21 Page 21 of 29
499
U/mg enzymec,d
Free ROL
Immobilization yieldb (%) ---
Silica-ROL
96
7.2
Silica-NH2-ROL
95
6.5
SBA-ROL
86
6.5
84
SBA-NH2-ROL
85
5.3
69
MCM-ROL
65
5.6
73
MCM-NH2-ROL
63
5.0
65
512 513
ip t
cr
us
84
M
Table1. Parameters of different ROL preparations.
a
Immobilizations were performed as described in the experimental section. Yield is defined as the percentage of the soluble enzyme that becomes attached to the support. c Experimental condition for activity measurement: 1.25 ml of 0.8 mM p-NPB and 0.01-0.1 mL of lipase solution in 25 mM sodium phosphate buffer at pH 7.0 and 25°C. d Specific activity (U/mg lipase) is expressed as micromole of substrate hydrolyzed per minute per mg of ROL. e Expressed yield is defined as actual activity of each biocatalyst per its expected activity.
d
b
te
501 502 503 504 505 506 507 508 509 510 511
94
Ac ce p
500
7.7
Expressed yielde (%) 100
an
Enzyme derivativea
23 Page 22 of 29
513 Table 2. Selective hydrolysis of fish oil using free and immobilized ROL a.
Activitya
Selectivityb
Activity
Selectivity
Free ROL
0.037
2.5
0.033
2.7
Silica-ROL
0.034
7.2
0.026
9.7
Silica-NH2-ROL
0.028
10.1
0.025
10.8
SBA-ROL
0.024
10.6
0.02
10.3
SBA-NH2-ROL
0.025
10.0
0.021
MCM-ROL
0.026
3.1
0.022
MCM-NH2 -ROL
0.021
5.7
pH 5, 4◦C Activity 0.028
2.8
0.020
13.0
0.016
11.9
0.016
12.8
12.3
0.014
13.5
3.6
0.016
4.7
5.3
0.015
4.9
us
0.015
a
Selectivity
te
d
M
Activity is expressed as micromoles of PUFA (EPA and DHA) released per minute and per milligram of ROL. b Selectivity is expressed as the ratio between released EPA and released DHA.
Ac ce p
520
an
Biocatalysts
515 516 517 518 519
pH 5, 25◦C
ip t
pH 7, 25◦C
cr
514
24 Page 23 of 29
520
Highlights: New amino groups were introduced to the surface of ROL via chemical amination.
522
Multipoint immobilization of ROL was performed on epoxy-functionalized supports.
523
Stability of ROL was greatly improved by multipoint covalent immobilization.
524
The soluble enzyme and immobilized derivatives discriminate between EPA and DHA.
525
Immobilization caused to improve the selectivity of ROL in fish oil hydrolysis.
ip t
521
cr
526
Ac ce p
te
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an
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527
25 Page 24 of 29
Ac
ce
pt
ed
M
an
us
cr
i
*Graphical Abstract (for review)
Page 25 of 29
Scheme 1
Scheme 1.
O
O
O , Triethylamine
O Si OH OH
Toluene, Reflux, 4h
O O O
ip t
O
O O O
Si
O
O
Si
cr
O OH OH OH OH OH OH OH
Ac
ce pt
ed
M
an
us
O
Page 26 of 29
Figure
Figure 1.
100
ip t
90 80 Silica-ROL
MCM-NH2-ROL SBA-NH2-ROL
cr
Silica-NH2-ROL
50 40 30
us
Free ROL
60
20 10
an
SBA-ROL
70
residual activity(%)
MCM-ROL
0 45 °C
50 °C
55 °C
Ac
ce pt
ed
M
temperature
Page 27 of 29
Figure 2
Figure 2.
100 90
Silica-NH2-ROL MCM-NH2-ROL SBA-NH2-ROL
60
cr
Free ROL
70
50 40 30
us
SBA-ROL
residual activity(%)
MCM-ROL
ip t
80
Silica-ROL
20 10
an
0
2-Propanol
Propanol
Ac
ce pt
ed
M
Dioxane
Page 28 of 29
Figure 3
ip t
Figure 3.
cr
110
Silica-ROL
us
Silica-NH2-ROL
SBA-ROL
an
90
80
70 0
1
2
3
M
residual activity (%)
100
4
5
SBA-NH2-ROL MCM-ROL MCM-NH2-ROL
6
Ac
ce pt
ed
run
Page 29 of 29
S1381-1177(15)00052-1 http://dx.doi.org/doi:10.1016/j.molcatb.2015.02.011 MOLCAB 3115
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
29-12-2014 17-2-2015 18-2-2015
Please cite this article as:
Chemical amination of Rhizopus oryzae lipase for multipoint covalent immobilization on epoxy-functionalized supports: modulation of stability and selectivity
5
Maryam Ashjaria,b, Mehdi Mohammadi*c, Rashid Badria,b
ip t
1 2 3 4
6 7
a
8
Iran.
9
b
us
cr
Department of Chemistry, College of Science, Ahvaz Branch, Islamic Azad University, Ahvaz,
Department of Chemistry, Khouzestan Science and Research Branch, Islamic Azad University,
Ahvaz, Iran.
11
c
12
National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O.Box:14965/161
13
Tehran, Iran.
an
10
M
Bioprocess Engineering Department, Institute of Industrial and Environmental Biotechnology,
d
14
te
15
17 18
Ac ce p
16
19
*
20
Phone: (+98) 21 44580461
21
Fax: (+98) 21 44580399
22
E-mail: [email protected]
Corresponding author
23 24 25 1 Page 1 of 29
Abstract
27
Multipoint covalent attachment of Rhizopus oryzae lipase (ROL) on epoxy-functionalized silica
28
and silica nanoparticles (MCM-41 and SBA-15) is reported. Multipoint immobilization of
29
enzymes on these supports usually performs by the reaction between several epoxy groups of the
30
support and several Lys residues on the external surface of the enzyme molecules at pH 10.
31
However, this standard immobilization procedure is unsuitable for ROL due to the low stability
32
of ROL at pH 10. Introducing new amino groups with lower pKb to the surface of ROL by using
33
chemical amination strategy permits immobilization of the enzyme at lower pH values.
34
Immobilization/stabilization of aminated ROL was performed in two steps. First the enzyme is
35
covalently immobilized at pH 7.0 and then the already immobilized enzyme is further incubated
36
at pH 9.2 to promote the formation of further covalent linkages between the immobilized ROL
37
and the support. The results showed higher thermal and co-solvent stability for immobilized
38
derivatives of aminated ROL compared to the results obtained for the derivatives of not-
39
aminated ROL and free ROL. Influence of the immobilization procedure on selectivity of the
40
immobilized preparations was studied in selective hydrolysis of fish oil at three different
41
conditions. The selectivity and reusability of ROL was greatly improved after immobilization.
42
All the derivatives discriminate between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-
43
4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA.
cr
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44
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26
45
Keywords: Epoxy supports, Fish oil, Rhizopus oryzae lipase, Multipoint covalent
46
immobilization, Silica nanoparticles
47 2 Page 2 of 29
1. Introduction
49
Lipases represent a class of enzymes with important roles in many essential physiological
50
processes [1]. They are also involved in a wide variety of applications in detergent and food
51
industries, leather industry, environmental management, cosmetics and perfume industry,
52
biomedical applications and biosensors [2-4], The lipase from Rhizopus Oryzae is a 1,3-specific
53
and moderately stable enzyme which is commercially available in both soluble and immobilized
54
form [5] . ROL has a molecular size of 32,000 Da and a pI of 7.6. It has 21 residues of aspartic
55
(12) and glutamic (9) groups which is higher than the number of lysine moieties (15 residues).
56
Regarding the high selectivity of ROL, several applications of this enzyme in hydrolysis and
57
esterification of fish oils were reported in literature [6, 7]. The beneficial health effects of fish oil
58
are now well documented and attributed to omega-3 polyunsaturated fatty acids (n-3 PUFA), cis-
59
5,8,11,15,17-eicosapentaenoic acid and cis-4,7,10,13,16,19-docosahexaenoic acid in particular
60
[8]. Due to the importance of polyunsaturated fatty acids, various techniques have been used for
61
enrichment of these compounds. One of the most promising techniques is the use of lipase-
62
catalyzed enzymatic hydrolysis of fish oil [9]. Lipases, both in soluble and immobilized form
63
discriminate against EPA and DHA; the main bioactive fatty acids in fish oil [10].
64
The improvement of efficiency of lipases in chemical reactions is still one of the main issues for
65
their effective application as industrial biocatalysts. Immobilization of lipase on solid supports is
66
the most known methods for such improvements [11]. Several attempts have been carried out on
67
the preparation of immobilized lipases, which involves a variety of immobilization techniques
68
and new support materials [12]. Enzyme immobilization permits to obtain a heterogeneous
69
catalyst, and if properly designed, improved stability and selectivity of the enzyme can be
70
expected.
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3 Page 3 of 29
The most of strategies for covalent immobilization of enzymes is based on using the amino
72
groups of the Lys residues which are the most abundant nucleophilic groups of the protein
73
surface [13]. The most obstacle of using these groups for immobilization of enzymes is that they
74
are reactive only at pH values over 10. Introducing new amino groups with lower pH values on
75
the protein surface via chemical amination of the carboxylic groups of Asp and Glu is a well
76
described method to optimize immobilization process [14]. In this way, the lower pKb of the new
77
amino groups allows to immobilize the enzyme via multipoint covalent attachment under milder
78
condition [15]. This type of irreversible immobilization is particularly preferred when the pH
79
stability of the used enzyme is relatively low. Moreover chemical amination increases the
80
number of interactions between enzyme and activated supports, promoting a higher number of
81
covalent attachments that increase enzyme stabilization.
82
Epoxy-activated supports are usually used to perform immobilization of proteins and enzymes at
83
mild conditions. These activated supports can be stored for a long time and have high stability at
84
neutral pH values [16]. At this condition epoxy groups can react with nucleophilic groups
85
present on the protein surface; in particular with terminal amino groups. However, it is well
86
documented that much stabilization of the enzyme could be expected if the immobilization
87
process occurs through several residues [17]. Immobilization of different enzymes on epoxy
88
supports via multipoint covalent attachment has been reported as an efficient way to improve the
89
enzyme stability [18].
90
chymotrypsin
91
immobilization/stabilization procedure [19]. To promote multipoint covalent attachment they
92
incubated the already immobilized enzyme under more drastic conditions (pH> 10 and long time
93
incubation). The results showed that the stability of the final derivatives was remarkably
Ac ce p
te
d
M
an
us
cr
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71
on
Mateo et al. reported immobilization of penicillin G acylase and
epoxy-activated
support
(Eupergit
C)
via
a
three-step
4 Page 4 of 29
increased by favoring the additional linkage between the immobilized enzyme molecule and the
95
support. Some other reports also pointed the positive effect of multipoint covalent attachment of
96
enzymes on epoxy supports to improve functional properties of the immobilized enzymes. To the
97
best of our knowledge, there is no report on immobilization of ROL on epoxy-functionalized
98
siliceous materials. The strategy of chemical amination to perform a stabilized–immobilized
99
derivative of ROL on epoxy-functionalized supports is also reported for the first time. To check
100
the effect of chemical amination-immobilization of ROL on its selectivity, we have used the
101
selective hydrolysis of fish oil in a biphasic system.
102
2. Materials and methods
103
2.1 Materials
104
The lipase from Rhizopus oryzae, fish oil from menhaden (containing 10-15% of
105
eicosapentaenoic
106
docosahexaenoic acid, ethylenediaminetetraacetic acid (EDTA), p-nitrophenyl butyrate (p-NPB),
107
sodium silicate, tetra ethyl ortho silicate (TEOS), polyuronic acid (P123), sodium periodate,
108
ethylenediamine
109
glycidoxypropyltrimethoxylsilane (GPTMS) were from Sigma. 1,4-dioxane, 1-propanol, 2-
110
propanol and silica gel (70 -230 mesh) were purchased from Merck. cis-5,8,11,14,17-
111
Eicosapentaenoic acid was from Cayman company. Other reagents and solvents were of
112
analytical or HPLC grade. Fourier transform infrared spectra (FT-IR) were recorded on a Bomen
113
FT-IR-MB-series instrument with a KBr pellet technique. Thermogravimetry (TGA) and
114
differential thermal analysis (DTA) were carried out from 10°C to 800 °C at a heating rate of 20
115
°C/min in air atmosphere using a STA 503M system from Bahr GmbH, Germany.
116
2.2. Methods
an
8-15%
M
and
of
docosahexaenoic
acid),
cis-4,7,10,13,16,19-
1-ethyl-3-(dimethylaminopropyl)
Ac ce p
(EDA),
te
d
acid
us
cr
ip t
94
carbodiimide
(EDC)
and
5 Page 5 of 29
2.2.1 Preparation of the silica nanoparticles
118
2.2.1.1 Preparation of SBA-15
119
Pure siliceous SBA-15 was prepared using a previously reported procedure [20]. Pluronic P123
120
triblock copolymer (EO20-PO70-EO20, BASF) was used as template. Briefly, four grams of
121
Pluronic P123 were added to 144 mL of an aqueous solution of HCl (2 M) at 40 °C.
122
Successively, TEOS was added dropwise (mass ratio TEOS/P123= 2:1) and then stirred for 2 h.
123
Afterwards, the mixture was transferred to Teflon-lined sealed container and kept at 100 °C for
124
48 h. The white solid was filtered, washed with distilled water and calcined at 550°C to remove
125
the template.
126
2.2.1.2 Preparation of MCM-41
127
Sodium silicate (19.0 g, 27% SiO2, diluted with 40.6 mL of distilled water) was mixed with a
128
solution of 16.4 g cetyltrimethylammonium bromide (CTAB) in 69.2 mL of distilled water. The
129
sodium silicate/CTAB solution was adjusted to pH 11.0 and stirred for 30 min. Finally the
130
mixture transferred into a stainless steel jacketed Teflon vessel and heated at 100 °C for 48 h.
131
Calcination of the obtained solid was carried out at 550 °C to remove the template [21].
132
2.2.2. Functionalization of silica, SBA-15 and MCM-41,
133
Surface modification of silica, SBA-15 and MCM-41 was performed according to the
134
literature[22]. Briefly, the dry mesoporous silica materials (1 g) were dispersed in 50 mL of dry
135
toluene; then 1-2 mL of GPTMS and 0.15 mL Et3N were added. The resulting mixture was
136
refluxed under nitrogen atmosphere and vigorous stirring for 4 h. The modified nanoparticles
137
were collected by filtration and washed thoroughly with THF. Finally the modified particles
138
were dried at 120 °C for 8 h.
139
2.2.2.1 Determination of epoxy groups on the support
Ac ce p
te
d
M
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ip t
117
6 Page 6 of 29
Determination of the epoxy groups on the support was carried out as follows: 200 mg of the
141
support was added to 1.5 mL of 1.3 M sodium thiosulphate solution and then the solution was
142
titrated by addition of 0.1 M hydrochloric acid until neutralization. The amount of epoxy groups
143
was calculated from the amount of hydrochloric acid needed to maintain neutrality of the
144
mixture. The same reaction was performed using unmodified nanoparticles as blank [23].
145
2.2.3. Chemical amination of ROL.
146
Chemical modification of the carboxyl groups of ROL was performed according to the method
147
described by Hoare and Koshland [24]. Briefly the amount of 300 µL of an aqueous solution of
148
ethylenediamine (1 M) was added to 10 mL of a solution containing ROL (1 mg/mL) in distilled
149
water. Subsequently to initialize the reaction 10 mM of 1-ethyl-3-(dimethylaminopropyl)
150
carbodiimide was added and the pH was adjusted to 4.7. After 3h the reaction mixture was
151
extensively dialyzed against water.
152
2.2.4. Enzymatic activity assay
153
The activities of the soluble lipase and its immobilized preparations were analyzed
154
spectrophotometrically by measuring the increment in absorbance at 348 nm (= 5150 M-1cm-1).
155
The increase in absorbance produced by the release of p-nitrophenol in the hydrolysis of p-NPB
156
in 25 mM sodium phosphate buffer at pH 7.0 and 25°C. Briefly, 0.01-0.1 mL of the lipase
157
suspension or solution (blank or supernatant without further dilution) was added to 1.25 mL of
158
substrate solution (0.8 mM) under magnetic stirring [15]. Spontaneous hydrolysis of p-NPB was
159
measured using 1.25 mL of substrate solution (0.8 mM) in the absence of ROL as control.
160
Enzymatic activity is given as 1µmol of p-nitrophenol released per minute per mg of the enzyme
161
(IU) under the condition described above.
162
2.2.5 Enzyme immobilization on epoxy-functionalized supports
Ac ce p
te
d
M
an
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ip t
140
7 Page 7 of 29
2.2.5.1 Immobilization of not-aminated ROL on epoxy-functionalized supports
164
50 mg of epoxy-functionalized SBA-15, MCM-41 and 300 mg of epoxy-functionalized silica
165
was incubated in 3 mL of 25 mM potassium phosphate buffer at pH 7.0 containing (1 mg/mL) of
166
ROL at 25 °C for 24 h. Periodically, samples of the supernatants were withdrawn and analyzed
167
for determination of the protein concentration by the Bradford's method [25]. Finally the
168
immobilized ROL derivatives were filtered and washed by distilled water and stored at 4°C.
169
2.2.5.2 Immobilization of aminated ROL on epoxy-functionalized supports
170
To perform the first immobilization step, 3 mL of aminated ROL (1 mg/mL) was offered to 50
171
mg of epoxy-functionalized SBA-15, MCM-41 and 300 mg of epoxy-functionalized silica at pH
172
7.0 at 25 °C. After immobilization of the soluble aminated enzyme (disappearance of hydrolytic
173
activity of the supernatant) the final pH of the solution was adjusted to 9.2 and the suspension
174
was incubated over night at 4°C under mild stirring. Finally the suspension was filtered and
175
washed with abundant distilled water.
176
2.2.5.3. Determination of the amount of protein bonded to the carriers
177
The amount of protein in supernatant and blank was determined by the Bradford method. The
178
amount of immobilized ROL was calculated by subtracting the amount of the enzyme in
179
supernatant from the total amount of the lipase used for immobilization. The reported yields of
180
immobilization were calculated as the ratio of the amount of the protein on the support to the
181
initial amount. Yields were expressed as percentage.
182
2.2.5.4. Leaching experiment
183
100 mg of each biocatalyst was incubated in a solution containing 1 M of NaCl with vigorous
184
magnetic stirring for 24 h. Then the concentration of ROL in the supernatant was measured by
185
the Bradford's method.
Ac ce p
te
d
M
an
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ip t
163
8 Page 8 of 29
2.2.6. Thermal inactivation of different ROL immobilized preparations
187
Free enzyme and immobilized preparations of ROL were incubated in 25 mM sodium phosphate
188
at pH 7.0 and different temperatures. The suspension of each sample was withdrawn periodically
189
and their activities were measured using the p-NPB assay.
190
2.2.7. Co-solvent stability of ROL and immobilized preparations
191
Free enzyme and immobilized preparations of ROL were incubated in a total volume of 1 mL
192
solution containing 25 mM sodium phosphate buffer pH 7.0 and 10% and 20% of 1,4-dioxane, 1-
193
propanol and isopropanol at 25°C. The suspension of each sample was periodically withdrawn
194
and their activities checked with the enzymatic activity assay as described above.
195
2.2.8. Hydrolysis of Fish Oil
196
Hydrolysis of fish oil was carried out in a biphasic system containing aqueous and organic
197
solvent [26]. 4.5 mL of cyclohexane, 500 µL of fish oil and 5mL of phosphate buffer (25 mM)
198
pH 5.0 and 7.0 were added in a test tube and pre-incubated at 25°C for 15 min with vigorous
199
stirring. To start the reaction, 50 mg of immobilized preparations or a solution containing 3 mg
200
of the free lipase were added to the reaction medium. Progress of the reaction was followed by
201
taking 100 µL of organic phase at selected time intervals followed by addition of 200 µL of 2-
202
propanol. Afterward, the selectivity and hydrolytic activity of each derivative were evaluated by
203
using the reverse-phase HPLC (Knauer with an UV detector) on a Grace C4 (25cm ×0.46cm).
204
The mobile phase was 55% of acetonitrile/45% of 10 mM ammonium phosphate (V/V) at pH 3.0
205
at flow rate of 0.4 mL/min and 210 nm in the UV detector. The retention times for the
206
unsaturated fatty acids were 25 and 29 min for EPA and DHA respectively. These enzymatically
207
produced PUFA were compared to their corresponding pure commercial standards.
208
2.2.9. Recyclability of immobilized derivatives
Ac ce p
te
d
M
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ip t
186
9 Page 9 of 29
The recyclability was studied by determining activity of immobilized derivatives in subsequent
210
reactions relative to that of the first reaction (pH 7.0 and 25°C). After each cycle (8h), enzyme
211
loaded particles were washed with cyclohexane and re-introduced into a fresh reaction medium
212
for another assay run and this procedure was repeated up to five cycles in the same condition.
cr
3. Result and Discussion 3.1. Preparation, functionalization and quantification of the supports
us
213 214 215 216 217
ip t
209
Siliceous materials have become a common choice of supports for enzyme immobilization.
219
Insolubility in water, high enzyme loading capacity, mechanical strength, high reactivity towards
220
functionalizing agents are the unique advantages of these materials. Their porous structure
221
creates a protective environment where the enzymes can tolerate more extreme pH, elevated
222
temperature and higher salt concentrations. Commercially available silica gel with moderate
223
surface areas (500 m2/g) and mesopuros silica nanoparticles (SBA-15, MCM-41) with ordered
224
pore structure were used for immobilization of ROL. Large-pore SBA-15 with surface area of
225
952 m2/g and pore sizes of 10.2 nm and MCM-41 with surface area of 1275 m2/g and pore sizes
226
of 3.9 nm were synthesized according to our previously published paper [27]. The epoxy-
227
functionalization
228
glycidoxypropyltrimethoxylsilane (Scheme 1). Quantification of oxirane groups on these
229
supports was performed by titration of the released hydroxide ion from the reaction between
230
epoxy groups on the support and sodium thiosulphate. The results revealed that the amount of
231
epoxy groups on the surface of silica, SBA-15 and MCM-41 was about 277, 805 and 850 µmol
232
per gram of each support, respectively. High degree of functionalization of SBA-15 and MCM-
233
41 compared to silica can be attributed to more surface area of these supports.
234
3.2. Multipoint covalent immobilization of ROL
Ac ce p
te
d
M
an
218
of
silica,
SBA-15
and
MCM-41
was
carried
out
using
3-
10 Page 10 of 29
Multipoint covalent attachment of enzymes on activated supports is a very powerful tool for
236
stabilizing proteins. The stabilization factor of the immobilized enzyme depends on the number
237
of linkages between support and enzyme. The formation of multiple covalent bonds, keeps the
238
relative positions of all the groups involved in immobilization unchanged during conformational
239
change induced by any distorting agent (heat, organic solvents, extreme pH values). This strategy
240
is usually used for immobilization of enzymes on aldehyde-functionalized supports [28].
241
However there are few reports of multipoint attachment of enzymes on epoxy-functionalized
242
supports [17-19]. These investigations are almost based on incubation of enzymes at high pH
243
values (pH>10) to promote several linkages between amino groups of Lys residues and the
244
epoxy groups of the support. Our investigation showed that ROL is clearly unstable at pH 10;
245
making impossible its multipoint covalent attachment on the epoxy-functionalized supports via
246
Lys residues. Therefore new amino groups with lower pkb were introduced to the surface of the
247
enzyme by using the chemical amination strategy. In this way ethylenediamine was used to
248
amination of Asp and Glu residues. The lower pkb of the new amino groups permits
249
immobilization of ROL at lower pH values, where the enzyme is completely stable. The
250
chemical amination of the protein surface was performed via the reaction between
251
ethylenediamine and carboxylic groups of the free lipase, after activation with carbodiimide. The
252
results showed that chemical amination has low impact on the enzyme activity; decreasing only
253
5-8% of its initial activity. The loss of 3-33% in enzyme activity after chemical amination of
254
penicillin G acylase and glutaryl acylase at different conditions have previously reported [29].
255
The aminated enzyme was immobilized on the supports via a two-step procedure. First most of
256
the enzyme was covalently immobilized under very mild experimental conditions (pH 7.0 and
257
25°C). The most reactive group at this condition is the terminal amino groups of the protein.
Ac ce p
te
d
M
an
us
cr
ip t
235
11 Page 11 of 29
Then the already immobilized enzyme was further incubated at pH 9.2 to facilitate the formation
259
of new covalent linkages between the immobilized enzyme molecule and the support. In this
260
way, all stabilizing advantages already achieved by the first immobilization process will be taken
261
to facilitate the long-term incubation of the enzyme derivative under hard experimental
262
condition. The immobilization of not-aminated ROL on each support was also performed at pH
263
7.0 and 25°C. As can be seen from table 1, almost complete immobilization of aminated and not-
264
aminated ROL on silica-epoxy achieved; producing 7.2 and 6.5 U/mg specific activity,
265
respectively. Compared to the specific activity of the soluble enzyme (7.7 U/mg enzyme), it
266
shows about 6-14% reduced activity. In order to perform immobilization on functionalized SBA-
267
15 and MCM-41, a solution containing 60 mg of ROL was offered to 1g of each support at low
268
ionic strength (25 mM). As can be seen in table 1, 85 and 86% of aminated and not-aminated
269
ROL is bound to SBA-epoxy after 24h of incubation respectively. The specific activity of
270
aminated ROL showed about 30% decrease after immobilization on this support. It seems that
271
the porous structure of SBA-15 and higher amount of immobilized enzyme compared to silica-
272
epoxy, increases diffusion limitation of substrate/product. Low immobilization yield (63-65 %)
273
was obtained in immobilization of aminated/not-aminated ROL on MCM-epoxy support. It is
274
most likely because of the fact that the small pore size (3.9 nm) of this support is not adequate to
275
make the internal surface accessible for immobilization of the enzyme. In other worlds, the
276
enzyme gets stuck in the pore entrances subsequently blocking the pore. The specific activity of
277
both aminated and not-aminated ROL decreased after immobilization on MCM-epoxy.
278
Generally there are many reports of decrease in enzyme activity after covalent immobilization on
279
different supports [30]. It can be resulted by some phenomena like denaturation of the protein
Ac ce p
te
d
M
an
us
cr
ip t
258
12 Page 12 of 29
during immobilization, altering microenvironment of the enzyme and diffusion limitation after
281
immobilization of enzyme.
282
In order to ensure the covalent attachment of ROL on the modified supports; leaching
283
experiments were performed. The condition of desorption of the protein from the supports was
284
examined by incubation of all the immobilized derivatives in a solution containing 1M NaCl at
285
25 °C for 24h. The activity of the supernatant was measured by p-NPB hydrolysis without
286
showing any measurable activity. The Bradford assay showed also no detectable enzyme in
287
solution clearly proving that the immobilization is exclusively performed via covalent binding.
288
3.3. Thermal stability of free and immobilized derivatives of ROL
289
Increased stability is one of the most common expected results of covalent immobilization of
290
enzymes and is often an important economic factor. Thermal stability of free ROL and the
291
immobilized derivatives was investigated at different temperatures. As figure 1 shows the
292
soluble enzyme is relatively unstable and loses 64, 82, 96% of its activity after 2h incubation at
293
45, 50 and 55 °C respectively. Rapid inactivation of the soluble ROL reveals the necessity of
294
using immobilization techniques to improve its stability. The results showed that immobilization
295
of non-modified ROL on silica-epoxy improved its thermal stability; remaining about 50% of its
296
initial activity after 2h incubation at 50 °C. Improved stability of ROL immobilized on MCM-
297
epoxy and SBA-epoxy compared to the free enzyme is also observed. These derivatives are
298
completely stable after 2h incubation at 45 °C. Figure 1 clearly shows the positive effect of
299
multipoint covalent attachment of enzyme on epoxy-functionalized supports. All the derivatives
300
are quite stable at 45 °C; keeping 100% of their initial activities after 2h of incubation. While the
301
free enzyme is almost inactive at 55 °C, silica-NH2-ROL, SBA-NH2-ROL and MCM-NH2-ROL
302
keeps 38%, 60% and 57% of their initial activities at the same condition, respectively. These
Ac ce p
te
d
M
an
us
cr
ip t
280
13 Page 13 of 29
higher stabilities of aminated ROL immobilized on epoxy supports are due to possibility of
304
multipoint covalent attachment with the epoxy groups, and the fact that in the used condition (pH
305
9.2) not only terminal amino groups may react with the supports, but also chemically introduced
306
amino groups.
307
3.4. Solvent stability of free and immobilized derivatives of ROL
308
The use of organic solvents for enzymatic reactions has demonstrated to be a useful method to
309
increase the efficiency of biocatalysts [31]. Most of the enzymes are usually not suited for
310
application in non-aqueous media in industrial processes. It has been reported that the polar
311
solvents can interact with the enzyme and reduce its catalytic activity [31]. This is due to the fact
312
that a few amounts of water molecules are required for enzymatic function. Organic solvents,
313
particularly those having log P values below 2 strongly distort this essential water-enzyme
314
interaction thereby inactivating the enzyme [32]. The use of immobilized form of enzymes is
315
considered as an efficient way to improve their stabilities in presence of organic solvents [33, 34]
316
The effect of immobilization on co-solvent stability of ROL was investigated in presence of three
317
water-miscible solvents (10% and 20% of 1-propanol, 2-propanol and dioxane) (Figure 2). These
318
polar organic solvents with log P<2 make a harsh condition to evaluate the stability of the
319
derivatives. The result showed that the soluble enzyme retains 72-91% of its initial activity after
320
24h of incubation in presence of 10% of each solvent. However, after increasing percentage of
321
the solvents to 20%, remarkable decrease in enzyme activity is observed especially for 1-
322
propanol. As figure 2 shows immobilization of ROL on the supports clearly improves its co-
323
solvent stability. SBA-ROL and MCM-ROL show slightly higher stabilities in presence of 10
324
and 20% of the solvents compared to silica-ROL. This might be explained by altering
325
microenvironment of the immobilized enzyme due to different nature of the micro and nano
Ac ce p
te
d
M
an
us
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ip t
303
14 Page 14 of 29
structure of the supports. However, limitations in substrate access, immobilization of the enzyme
327
from different side and unfavourable enzyme conformation may also be contributing factors. The
328
most promising results obtained from the investigation of the stability of aminated ROL
329
immobilized on different supports. As can be concluded from figure 2, chemical amination
330
greatly improves the stability of immobilized derivatives. SBA-NH2-ROL retains its whole
331
activity after 24h incubation in presence of 10 and 20% of each solvent. These results suggest
332
high ability of chemical amination approach to produce derivatives suitable to use in chemical
333
reactions in which presence of organic solvents and long time of reaction are needed.
334
3.5. Fish oil hydrolysis
335
It has been reported that chemical amination and subsequent immobilization of the aminated
336
enzymes leads to modulate their catalytic properties (selectivity and activity) [14]. Alterations of
337
the shape and size of the active centre has been proposed as a possible cause of such modulation.
338
By using free and immobilized derivatives of ROL the hydrolysis of fish oil from menhaden in a
339
biphasic system containing aqueous and organic solvent was studied. HPLC-UV analysis was
340
used to follow the rate of PUFAs release and the selectivity of the preparations against EPA and
341
DHA as the most valuable ingredients of the oil. The Selectivity is calculated as the ratio
342
between released EPA and released DHA and activity was calculated by the following equation:
an
M
d
te
Ac ce p
Activity =
343
us
cr
ip t
326
poly unsaturated fatty acid concentration (mmol) enzyme (mg) × time (minute)
344
As same as the results obtained in p-NPB reaction (table 1) covalent immobilization of ROL
345
causes to decrease in enzyme activity. The derivatives obtained from immobilization of
346
chemically aminated enzyme produce lower activities compared to the not-aminated
347
preparations. Between the three selected conditions for the hydrolysis reaction, pH 7.0 and 25 °C
348
were the optimal condition in terms of enzymatic activity. Beside of activity, the EPA/DHA 15 Page 15 of 29
selectivity of the used biocatalyst is also a critical parameter in fish oil hydrolysis. As EPA and
350
DHA are very similar and difficult to be separated by using physico-chemical protocols,
351
selective hydrolysis of fish oil can be a powerful to produce almost pure EPA. As can be seen in
352
table 2, while the free enzyme poorly discriminates between EPA and DHA, its selectivity
353
greatly improves after immobilization both in aminated and not-aminated forms. Wide range of
354
selectivities (3.1-13.5) were produced depends on the condition of reaction and the kind of
355
procedure/support used for immobilization. All the derivatives were also observed to display a
356
significant preference for EPA as compared to DHA which is in accordance with previous
357
reports [10, 26]. As the results show, lowering the temperature causes to significant improvement
358
in enzyme selectivity. Moreover the derivatives of chemically aminated ROL shows higher
359
selectivity compared to not-aminated ROL preparations. While the selectivity of the soluble
360
ROL is 2.8 at pH 5.0 and 4°C, SBA-NH2-ROL shows almost 5 fold improvement in selectivity at
361
the same condition. The observed selectivity of this derivative permits the production of PUFA
362
with almost 93 % of EPA purity at the first stages of the reaction. In a previously published
363
report Fernandez et al. have investigated on EPA/DHA selectivity of 7 different lipases
364
immobilized on octyl-sepharose and cyanogen bromide-sepharose. The obtained selectivities
365
(6.9 and 9.8 for octyl-ROL and CNBr-ROL, respectively) were the best results of the examined
366
biocatalysts [7].
367
Insignificant improvement in selectivity was observed for the derivatives obtained from
368
immobilization of both aminated and not-aminated ROL on epoxy-functionalized MCM-
369
41(MCM-ROL and MCM-NH2-ROL). This observation clearly demonstrates that apart from the
370
reaction condition and modification of the enzyme surface, the size and shape of the support can
371
influence the catalytic properties of the enzyme. Regarding the activity and selectivity of the
Ac ce p
te
d
M
an
us
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ip t
349
16 Page 16 of 29
derivatives, the best results were obtained for silica-ROL with 13.5 selectivity and 0.02 (U/mg
373
lipase per minute) at pH 5.0 and 4 °C.
374
3.3.5. Recyclability of immobilized derivatives
375
One of the technological and economical advantages of enzyme immobilization is the ability of
376
the immobilized preparations to repeatedly use in chemical reactions. In order to investigate
377
reusability of the immobilized derivatives they were used in hydrolysis reaction for five cycles.
378
After each run (8 h), the immobilized preparations were recovered by filtration, washed with
379
cyclohexane and reused for a new reaction under the same conditions. The activity of first cycle
380
of the reaction was set as 100% and the activity in the subsequent reactions was calculated
381
accordingly. As figure 3 shows the derivatives retain about 81-94% of their activities after five
382
cycles of the reaction. The results also present a meaningful improvement of recyclability of the
383
three derivatives of aminated ROL confirming that chemical amination has positive effect on
384
enzymatic function of ROL. The best result of reusability was obtained for SBA-NH2-ROL with
385
97% retaining of its activity after five cycles of the reaction.
cr
us
an
M
d
te
Ac ce p
386
ip t
372
387
4. Conclusion
388
Covalent immobilization of enzymes on epoxy-functionalized supports is normally carried out
389
under very mild conditions. Under these conditions the intense multipoint covalent linkage
390
between the immobilized enzyme and the support is not possible. This is due to the low
391
reactivity of the epoxy groups and the reactive groups of the protein at pH 7. Therefore
392
producing an effective strategy to broaden thermal and co-solvent stability and selectivity of
393
ROL was considerable target of this research. For this purpose, chemical amination of ROL and
394
immobilization of aminated/not-aminated ROL on different epoxy-functionalized supports were
17 Page 17 of 29
performed. Chemical amination introduces new amino groups with a lower pKb value than that
396
of the Lys residues. The derivatives of the chemically aminated ROL are more stable than
397
derivatives of the not-aminated ROL with respect to thermal and solvent inactivation. The most
398
interesting results were found for SBA-NH2 –ROL, which showed retaining of 100% of its initial
399
activity after 24h incubation in presence of 20% of 1-propanol, 2-propanol and dioxane. This
400
derivative also showed higher thermal stability compared to other derivatives. Beside of these
401
interesting results, the selectivity of the enzyme was also modulated using chemical amination
402
procedure. Although the selectivity of the derivatives from MCM-epoxy is relatively low, most
403
of the derivatives showed high selectivity in fish oil hydrolysis compared to free enzyme. The
404
results demonstrate that, for the development of an optimal catalyst for the production of omega-
405
3 fatty acids, it is necessary to consider factors such as the immobilization protocol and the kind
406
of support. Also remarkable improvement in selectivity and stability of the immobilized
407
derivatives compensates undesirable decrement of activity during chemical amination and
408
covalent immobilization of ROL.
cr
us
an
M
d
te
Ac ce p
409
ip t
395
410
Acknowledgment
411
This work was financially supported by the Iran National Science Foundation (INSF) (grant
412
number 91004274) for which the authors are thankful.
413 414 415 416 417 418
18 Page 18 of 29
419
References
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[17] V. Grazú, O. Abian, C. Mateo, F. Batista‐Viera, R. Fernández‐Lafuente, J.M. Guisán,
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Biotechnology and bioengineering, 90 (2005) 597-605. [18] F. López-Gallego, L. Betancor, A. Hidalgo, C. Mateo, J.M. Guisán, R. Fernández-Lafuente, Journal of biotechnology, 111 (2004) 219-227. [19] C. Mateo, O. Abian, R. Fernandez–Lafuente, J.M. Guisan, Enzyme and Microbial Technology, 26 (2000) 509-515.
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[20] A. Salis, M. Pisano, M. Monduzzi, V. Solinas, E. Sanjust, Journal of Molecular Catalysis B: Enzymatic, 58 (2009) 175-180. [21] K.A. Northcott, K. Miyakawa, S. Oshima, Y. Komatsu, J.M. Perera, G.W. Stevens, Chemical Engineering Journal, 157 (2010) 25-28. [22] J. Lin, J.A. Siddiqui, R.M. Ottenbrite, Polymers for Advanced Technologies, 12 (2001) 285292. [23] L. Sundberg, J. Porath, Journal of Chromatography A, 90 (1974) 87-98. [24] D.t. Hoare, D. Koshland, Journal of Biological Chemistry, 242 (1967) 2447-2453. [25] M.M. Bradford, Analytical biochemistry, 72 (1976) 248-254. [26] M. Mohammadi, Z. Habibi, S. Dezvarei, M. Yousefi, Food and Bioproducts Processing, (2014). [27] M. Mohammadi, Z. Habibi, S. Dezvarei, M. Yousefi, S. Samadi, M. Ashjari, Process Biochemistry, 49 (2014) 1314-1323. [28] R.C. Rodrigues, C.A. Godoy, G. Volpato, M.A. Ayub, R. Fernandez-Lafuente, J.M. Guisan, Process Biochemistry, 44 (2009) 963-968. [29] F. López-Gallego, T. Montes, M. Fuentes, N. Alonso, V. Grazu, L. Betancor, J.M. Guisán, R. Fernández-Lafuente, Journal of biotechnology, 116 (2005) 1-10. [30] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente, Enzyme and Microbial Technology, 40 (2007) 1451-1463. [31] F.H. Arnold, Trends in biotechnology, 8 (1990) 244-249. [32] A.M. Klibanov, Nature, 409 (2001) 241-246. [33] R. Fernandez-Lafuente, C. Rosell, L. Caanan-Haden, L. Rodes, J. Guisan, Enzyme and Microbial Technology, 24 (1999) 96-103. [34] V. Stepankova, S. Bidmanova, T. Koudelakova, Z. Prokop, R. Chaloupkova, J. Damborsky, ACS Catalysis, 3 (2013) 2823-2836.
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20 Page 20 of 29
Figure captions:
484
Scheme 1. A description of the supports treatment used for immobilization.
485
Figure 1. Thermal stability of free ROL and immobilized preparations at 45°C, 50 °C, 55 °C.
486
Experimental condition: Increasing certain amount of each biocatalyst in 1mL of sodium
487
phosphate buffer 25 mM (pH 7.0) and incubation at different temperatures for 2h. Initial activity
488
of ROL and each immobilized derivatives was determined in 1 mL of sodium phosphate buffer
489
25 mM (pH 7.0) at 25 °C and set as 100%.
490
Figure 2. Co-solvent stability of free ROL and immobilized preparations in presence of 20 % of
491
organic solvents. Experimental condition: Incubation of each biocatalyst in 1 mL solution
492
containing 25 mM sodium phosphate buffer (pH 7.0) and 20% of three organic solvents at 25°C.
493
Initial activity of ROL and each immobilized derivatives was determined in 1 mL of sodium
494
phosphate buffer 25 mM (pH 7.0) at 25 °C and set as 100%.
495
Figure 3. Effect of repeated use of immobilized preparations on their activity in fish oil
496
hydrolysis. Reaction conditions: A biphasic system containing 4.5 mL of cyclohexane, 5mL (25
497
mM) of phosphate buffer (pH 7, 25°C), 500 µL of fish oil and 100 mg of biocatalyst.
cr
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21 Page 21 of 29
499
U/mg enzymec,d
Free ROL
Immobilization yieldb (%) ---
Silica-ROL
96
7.2
Silica-NH2-ROL
95
6.5
SBA-ROL
86
6.5
84
SBA-NH2-ROL
85
5.3
69
MCM-ROL
65
5.6
73
MCM-NH2-ROL
63
5.0
65
512 513
ip t
cr
us
84
M
Table1. Parameters of different ROL preparations.
a
Immobilizations were performed as described in the experimental section. Yield is defined as the percentage of the soluble enzyme that becomes attached to the support. c Experimental condition for activity measurement: 1.25 ml of 0.8 mM p-NPB and 0.01-0.1 mL of lipase solution in 25 mM sodium phosphate buffer at pH 7.0 and 25°C. d Specific activity (U/mg lipase) is expressed as micromole of substrate hydrolyzed per minute per mg of ROL. e Expressed yield is defined as actual activity of each biocatalyst per its expected activity.
d
b
te
501 502 503 504 505 506 507 508 509 510 511
94
Ac ce p
500
7.7
Expressed yielde (%) 100
an
Enzyme derivativea
23 Page 22 of 29
513 Table 2. Selective hydrolysis of fish oil using free and immobilized ROL a.
Activitya
Selectivityb
Activity
Selectivity
Free ROL
0.037
2.5
0.033
2.7
Silica-ROL
0.034
7.2
0.026
9.7
Silica-NH2-ROL
0.028
10.1
0.025
10.8
SBA-ROL
0.024
10.6
0.02
10.3
SBA-NH2-ROL
0.025
10.0
0.021
MCM-ROL
0.026
3.1
0.022
MCM-NH2 -ROL
0.021
5.7
pH 5, 4◦C Activity 0.028
2.8
0.020
13.0
0.016
11.9
0.016
12.8
12.3
0.014
13.5
3.6
0.016
4.7
5.3
0.015
4.9
us
0.015
a
Selectivity
te
d
M
Activity is expressed as micromoles of PUFA (EPA and DHA) released per minute and per milligram of ROL. b Selectivity is expressed as the ratio between released EPA and released DHA.
Ac ce p
520
an
Biocatalysts
515 516 517 518 519
pH 5, 25◦C
ip t
pH 7, 25◦C
cr
514
24 Page 23 of 29
520
Highlights: New amino groups were introduced to the surface of ROL via chemical amination.
522
Multipoint immobilization of ROL was performed on epoxy-functionalized supports.
523
Stability of ROL was greatly improved by multipoint covalent immobilization.
524
The soluble enzyme and immobilized derivatives discriminate between EPA and DHA.
525
Immobilization caused to improve the selectivity of ROL in fish oil hydrolysis.
ip t
521
cr
526
Ac ce p
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M
an
us
527
25 Page 24 of 29
Ac
ce
pt
ed
M
an
us
cr
i
*Graphical Abstract (for review)
Page 25 of 29
Scheme 1
Scheme 1.
O
O
O , Triethylamine
O Si OH OH
Toluene, Reflux, 4h
O O O
ip t
O
O O O
Si
O
O
Si
cr
O OH OH OH OH OH OH OH
Ac
ce pt
ed
M
an
us
O
Page 26 of 29
Figure
Figure 1.
100
ip t
90 80 Silica-ROL
MCM-NH2-ROL SBA-NH2-ROL
cr
Silica-NH2-ROL
50 40 30
us
Free ROL
60
20 10
an
SBA-ROL
70
residual activity(%)
MCM-ROL
0 45 °C
50 °C
55 °C
Ac
ce pt
ed
M
temperature
Page 27 of 29
Figure 2
Figure 2.
100 90
Silica-NH2-ROL MCM-NH2-ROL SBA-NH2-ROL
60
cr
Free ROL
70
50 40 30
us
SBA-ROL
residual activity(%)
MCM-ROL
ip t
80
Silica-ROL
20 10
an
0
2-Propanol
Propanol
Ac
ce pt
ed
M
Dioxane
Page 28 of 29
Figure 3
ip t
Figure 3.
cr
110
Silica-ROL
us
Silica-NH2-ROL
SBA-ROL
an
90
80
70 0
1
2
3
M
residual activity (%)
100
4
5
SBA-NH2-ROL MCM-ROL MCM-NH2-ROL
6
Ac
ce pt
ed
run
Page 29 of 29