Subscriber access provided by Stony Brook University | University Libraries
Biotechnology and Biological Transformations
#-Amylase@magnetite: magnetic nanocomposites with enhanced thermal stability for starch hydrolysis Bazhena V. Astafyeva, Olga E. Shapovalova, Andrey S. Drozdov, and Vladimir V. Vinogradov J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01298 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 19
Journal of Agricultural and Food Chemistry
α-Amylase@ferria: magnetic nanocomposites with enhanced thermal stability for starch hydrolysis Bazhena V. Astafyeva, Olga E. Shapovalova, Andrey S. Drozdov,∗ and Vladimir V. Vinogradov Laboratory of Solution Chemistry of Advanced Materials and Technologies, ITMO University, St. Petersburg, 191002, Russian Federation E-mail:
[email protected] 1
Abstract
2
The present study is devoted to the development of a new class recyclable magnetic
3
catalytic nanocomposites for starch hydrolysis. Alpha-amylase was entrapped within
4
magnetite derived xerogel matrix in a course of a room-temperature sol-gel transition,
5
leading to enzyme immobilization within the pores of a rigid magnetic matrix. For
6
hybrid organo-inorganic composites with enzyme mass fractions less than 10 wt. %
7
no enzyme leaching was observed. At 80◦ C amylase@ferria composite demonstrates
8
catalytic activity on the level of 10 U/mg and starch hydrolysis rate comparable to
9
free enzyme, while at 90◦ C the activity of amylase@ferria at least twice higher that
10
of free amylase due to higher thermal stability of the composite. Entrapped amylase
11
showed excellent stability and lost only 9 % of its activity after 21 days of storage in a
12
buffer solution, while free enzyme was totally inactivated after 17 days. The material
13
can be used either a magnetically-separable reusable catalyst or can be applied as a
14
catalytic ceramic coating with at least 10 cycles of use.
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
15
Introduction
16
Advance in nanomaterials synthesis and nanotechnology led to active implementation of
17
nanostructured materials in different areas of industry as adsorbents, catalysts or construc-
18
tion materials. 1–4 When it comes to food industry, the main attention is paid to enzyme
19
immobilization, as it can significantly reduce costs of enzymatically catalyzed reactions by
20
thermal and chemical stabilization of enzymes, simplifying separation of enzymes from reac-
21
tion mixtures and offering the possibility to make recyclable enzymatic catalysts. 5–8 Selection
22
of a support material can have a critical effect on the stability of enzymes and the efficiency
23
of enzyme immobilization, although it is difficult to predict in advance which support will be
24
the most suitable for a particular enzyme. The support must be insoluble in water, should
25
have high enzyme loading capacity, be mechanically stable and must not have deleterious
26
effect on the catalytic activity of the enzyme. The variety of nanostructured materials for
27
immobilization of enzymes includes both organic and inorganic polymers and materials, such
28
as: porous polymeric matrixes, 9,10 polystyrene particles, 8,11,12 phospholipid liposomes, 13–15
29
zeolites, 16,17 metal oxides, 18,19 silica matrixes 20–22 and ect. Among all enzymatic nanocom-
30
posites significant attention is paid to ones with magnetic properties. 23,24 Magnetic field
31
susceptibility revealed a mechanism for efficient recovery of the enzyme complex thereby
32
preventing the contamination of the final product by enzymatic catalyst. Magnetic enzy-
33
matic catalysts mainly presented by either core-shell type structures or polymeric matrixes
34
with encapsulated magnetic nanoparticles and are known to be effective systems with good
35
application results. 25–30 Despite the effectiveness, magnetic composites shares one common
36
disadvantage of their complex structure that involves a magnetic core, an enzyme and a
37
linking agent. Such multicomponent structure requires multi-step synthesis procedures with
38
intermediate purification steps resulting in higher production costs that can negate the prac-
39
tical advantage of such systems. In this article, we describe magnetic nanocomposite with
40
amylase activity consisting of only two components: amylase and magnetite ceramic matrix.
41
The material is produced by direct entrapment of the enzyme within magnetite xerogel in a 2
ACS Paragon Plus Environment
Page 2 of 19
Page 3 of 19
Journal of Agricultural and Food Chemistry
42
course of the room-temperature sol-gel transition. Entrapped amylase not only demonstrated
43
the ability to perform catalytic hydrolysis of starch solutions with the rate comparable to that
44
of the free enzyme, but also showed excellent thermal stability and extended temperature
45
of activity window and prolonged period of storage. Composites demonstrated leach-proof
46
behavior and can be magnetically separated and reused up to 10 times with only 5% decrease
47
of its initial activity. Alternatively the composite can be applied in a form of ceramic coating
48
to ensure effective application in batch or flow reactors.
49
Materials and methods
50
Chemicals: Iron (II) chloride tetrahydrate, iron (III) chloride hexahydrate, ammonia 25%
51
solution, α-amylase from porcine pancreas (100 units/mg) and soluble starch, all obtained
52
from Sigma-Aldrich.
53
Preparation of a ferria hydrosol: Pure ferria hydrosol was prepared from iron (II) chloride
54
tetrahydrate and iron (III) chloride hexahydrate as described in the Ref. 31. Briefly, 2.5
55
g FeCl2 *4H2 O and 5 g FeCl3 *6H2 O were dissolved in 100 mL of deionized water under
56
constant stirring (500 rpm). Then, 12 mL of aqueous ammonia solution was added at room
57
temperature. Using a magnet, the formed magnetite precipitate was collected and washed
58
with deionized water until neutral pH. The washed black precipitate was mixed with 100 mL
59
of deionized water and subjected to ultrasonic treatment (37 kHz, 110 W) under constant
60
stirring (300 rpm) for 120 minutes. The mass fraction of magnetite nanoparticles in the
61
resulting sol was 2 wt. %.
62
Entrapment of α-amylase in a ferria matrix: 20 mL of freshly prepared ferria sol was
63
mixed with 0 to 1250 µL of α-amylase solution (4000 U/mL). After stirring, the contents
64
were dried in a vacuum desiccator to yield composites with enzyme mass fractions ranging
65
from 0 to 12.5 wt. % (0-11 U/mg). The composite materials were milled in an agate mortar
66
and used in experiments. To evaluate the release profile of the enzyme, 20 mg of the crushed
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
67
composite in a quartz cuvette was treated with 2 mL of the saline solution and the absorption
68
spectrum at 37◦ was measured over time at 210 nm.
69
Amylase@ferria coatings: In order to prepare the catalytic coating mixture of 5 mL of
70
freshly prepared ferria sol with 1 mL of α-amylase solution (4000 U/mL) was applied on
71
the walls of a glass and condensed on air. The glass was continuously rotated to ensure the
72
homogeneity of the coating.
73
Preparation of the starch solution: 2 g of soluble starch were placed in 98 mL of water
74
preheated to 55◦ and thoroughly stirred. The temperature was gradually elevated to 80◦ with
75
continuous stirring using a magnetic stirrer for an hour. After incubation the temperature
76
of the solution was decreased to 60◦ , it was stirred for another 30 minutes and used in the
77
experiments.
78
Evaluation of amylase@ferria particles enzymatic activity: Activity of the amylase@ferria
79
composite was evaluated by measuring the viscosity of the starch solution. 7 mL of the starch
80
solution was placed into a thermostated rotary viscometer sleeve and, after adding 100 mg
81
of the 10 wt. % amylase@ferria composite (enzymatic activity 10 U/mg), the viscosity
82
was measured in the kinetic mode at different temperatures ranging from 60 to 90◦ . For
83
comparison, 7 mL of the starch solution was incubated with 25 µL of amylase solution (4000
84
U/mL) and subsequently tested in the same manner.
85
Evaluation of amylase@ferria particles reusability: In order to test the reusability of the
86
composite magnetic catalyst 100 mg of the 10 wt. % amylase@ferria composite (enzymatic
87
activity 10 U/mg) was added to 7 mL of the starch solution and stirred for 20 minutes while
88
monitoring the process by measuring the viscosity of the solution. After that, the composite
89
material was separated by applying magnetic field and added to a new portion of the starch
90
solution for another catalytic cycle.
91
Catalytic activity of amylase@ferria coatings: Into a model reactor with the total volume
92
1 L, coated with amylase@ferria nanocomposite, 100 mL of the starch solution was added
93
and mixed at 80◦ for 15 minutes. The process was monitored by measuring the viscosity.
4
ACS Paragon Plus Environment
Page 4 of 19
Page 5 of 19
Journal of Agricultural and Food Chemistry
94
After the incubation, the solution was changed with a new portion of the starch solution and
95
another catalytic cycle was performed.
96
Characterization methods: The crystalline phase of the samples was measured by X-
97
ray diffraction (Bruker D8 Advance) using Cu-Kα radiation (λ = 1.54 ˚ A); the samples
98
were scanned at a rate of 0.5 degrees per minute. To analyze the samples with a high-
99
resolution scanning electron microscopy (SEM), the obtained ground xerogel was deposited
100
on a metal tip and investigated without additional spraying using a Magellan 400L ultra-high
101
resolution electron microscope. The samples for a transmission electron microscopy (TEM)
102
were obtained by dispersing a small probe in ethanol to form a homogeneous suspension.
103
Then, a suspension drop was coated on a copper mesh covered with carbon for a TEM
104
analysis (FEI TECNAI G2 F20, at an operating voltage of 200 kV). Specific surface area,
105
pore volume and pore size distribution were investigated using Quantachrome Nova 1200e by
106
nitrogen adsorption at 77 K and analyzed by the BET and BJH equations. Prior to analysis,
107
all samples were degassed at room temperature for 48 hours. Hydrodynamic diameter was
108
measured by the DLS technique on Photocor Compact Z. The Raman spectra were recorded
109
using the 633 nm He-Ne laser line on a Horiba Jobin Yvon Micro Raman 300 spectrometer.
110
The laser power on the samples employed was 0.03 mW and 0.344 mW, with 300 s and 120
111
s exposition per diffraction window, respectively. In all the measurements, 50x Olympus
112
lens, hole of 500mm, slit of 100mm and a diffraction grid with 1800 grooves/mm were
113
employed. The IR measurements were performed in transmission and ATR modes. Viscosity
114
was determined by a Fungilab rotational viscometer.
115
Results and discussion
116
Synthesis of amylase@ferria composites
117
In order to produce amylase@magnetite composites, the stable magnetite hydrosol (desig-
118
nated as ferria) was used that was described in details in our previous works. 31 The hydrosol 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
119
was synthesized by ultrasound-assisted coprecipitation procedure using non-stoichiometric
120
ratios of iron(II) and iron(III) salts with the subsequent ultrasonic treatment of the resulting
121
sol in deionized water. The final hydrosol consists of magnetite nanoparticles with an aver-
122
age hydrodynamic diameter of 30 nm dispersed in water at neutral pH level (pH 7). Surface
123
of the particles is densely covered with the hydroxyl groups both of magnetite and Fe(OH)2
124
nature as proven by Raman spectroscopy (Fig. 1a). Due to this fact, the isoelectric point
125
of the magnetite NPs is shifted from pH = 6.8, which is common for magnetite, to a higher
126
pH value of 8.3 (Fig. 1b). As a result, the magnetite NPs showed high zeta-potential values
127
(+36 mV at neutral pH level) and excellent colloidal stability, forming stable hydrosols which
128
are behaves as typical magnetic fluids (Fig. 1c).
Figure 1: Ferria hydrosol. The ATR spectra demonstrates the presence of the hydroxyl groups of both Fe(OH)2 and Fe3 O4 nature (a); in contrast to magnetite, the isoelectric point of the ferria nanoparticles is shifted to pH = 8.3 (b); due to the high zeta potential values the ferria nanoparticles form a stable hydrosol with a magnetic fluid-like behavior at a neutral pH level (c).
129
Amylase@ferria composites were produced by the condensation of the ferria hydrosol
130
with α-amylase solution under reduced pressure at room temperature. Upon removal of
131
the solvent, a sol-gel transition occured leading to the formation of the mesoporous xerogel
132
matrix. The matrix was composed of the 10 nm magnetite nanoparticles with a truncated
133
octahedron morphology according to SEM and TEM images (Fig. 2a and 2b). The XRD
134
pattern of the material demonstrated the lines typical for magnetite (compared to JCPDS
135
file no. 19-0629) (Fig. 2c). The main peak at 35.45◦ attributed to the crystalline plane with 6
ACS Paragon Plus Environment
Page 6 of 19
Page 7 of 19
Journal of Agricultural and Food Chemistry
136
Miller indices of (3 1 1) is clearly seen. Other distinctive peaks at 18.52◦ - (0 2 0), 30.10◦ -
137
(2 2 0), 43.08◦ - (4 0 0), 53.45◦ - (4 2 2), 56.98◦ - (5 1 1), 62.57◦ - (4 4 0), 70.99◦ - (5 3 3),
138
and 74.02◦ - (4 4 4) (Fig. 2f) matched those of the standard magnetite diffraction pattern. 32
139
Another proof that the ferria xerogel matrix consisted of the magnetite nanoparticles came
140
from Raman spectroscopy. The Raman spectra of the sample showed the characteristic
141
broad band of magnetite at 667 cm−1 (Fig. 2d, line a) when the spectra was recorded with
142
a 633 nm He-Ne laser (0.030 mW laser beam power and an acquisition time of 300 sec per
143
diffraction window). 33,34 Further confirmation came from observations of the known phase
144
transformations of magnetite under the Raman-laser measurement conditions: when the
145
power was increased to 0.344 and 1.35 mW (acquisition times of 90 and 30 sec, respectively),
146
then for the former the characteristic maghemite bands at 711, 492, and 372 cm−1 are seen
147
(Fig. 2d, line b), and for the latter full oxidation to α-hematite is observed (Fig. 2d, line c)
148
by the characteristic bands at 591, 385, 273, and 213 cm−1 . 33,34
149
The formed matrix possesses well-developed mesoporous nanoarchitecture (Fig. 2a,b)
150
with a high specific surface area (110 m2 /g for 10 wt. % amylase@ferria and 119 m2 /g
151
for pure ferria xerogel) and an average pore diameter of 8 nm (Fig. 1S(a,b)). While the
152
molecule of amylase has the size of 7x6x5 nm, 35 it is complementary to the pores diameter
153
of ferria matrix, and it is entrapped in the course of a sol-gel transition. The mechanism
154
of the entrapment can be briefly described as follows: at the first step, negatively charged
155
amylase molecules (isoelectric point of amylase is at pH = 5.5) 35 electrostatically interacts
156
with positively charged magnetite nanoparticles (isoelectric point of magnetite sol is at pH
157
= 8, Fig. 1b). Upon drying of the resulting mixture, the rigid magnetite xerogel matrix is
158
formed (Fig. 2a,b), in which enzyme molecules may be buried inside of the matrix or located
159
in the surface layer. According to our previous observations, such structural organization
160
of a material allows the entrapped enzyme to interact with macromolecular substrates, such
161
as polysaccharides and to perform enzymatic transformations without being released from
162
a xerogel matrix. 36,37 Due to the formation of interparticle Fe-O-Fe bonds in the process of
7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 2: The ferria xerogel matrix. The ferria matrix consists of nanoparticles with a truncated tetrahedron morphology. Interplanar spacing is shown in the insert (a); SEM image of the ferria xerogel demonstrates porous structure of the material (b); XRD patternt of the ferria xerogel shows peaks typical for the magnetite crystal phase (c); Raman spectra recorded at a 0.03 mW laser beam power demonstrates spectra typical for magnetite (line a), after increasing the laser power to 0.344 mW magnetite partially converts into maghemite (line b), at 1.35 mW complete conversion of magnetite into hematite is observed (line c). All lines are obtained from a single sample (d); the magnetization curve of ferria and amylase@ferria. Absence of hysteresis can be observed in the insert (e); the material is readily attracts to the magnet (f). 163
sol-gel transition, the formed composite matrix is not prone to spontaneous re-suspendeition
164
in water media and can be used as a magnetically-sensitive catalytic composite. 38 The mate-
165
rial demonstrated the typical superparamagnetic behavior: its magnetization curve showed
166
absence of the hysteresis loop and no remnant magnetization in absence of magnetic field
167
(Fig. 2e). The magnetic moment of the composite at 6000 Oe amounted to 61 emu/g for
168
10% amylase@ferria, that is 25% lower than for the pure ferria matrix. Nevertheless, a high
169
magnetic susceptibility allowed to separate the amylase@ferria composite using an external
170
magnet (Fig. 2f). The entrapment of amylase may be either full or there may be partial
171
release from the composite depending on the mass fraction of the enzyme. According to 8
ACS Paragon Plus Environment
Page 8 of 19
Page 9 of 19
Journal of Agricultural and Food Chemistry
172
release studies, at mass fractions of entrapped amylase below 10 wt. %, only a minor release
173
of approximately 1% of the total entrapped enzyme amount occurred, while at higher mass
174
fractions release was increased up to 21% of the total amount due to matrix enzyme loading
175
capacity overload (Fig. 2S). 36
176
Catalytic activity of amylase@ferria
177
To evaluate the enzymatic activity of the synthetized biocomposites, the enzymatic starch
178
cleavage reaction was selected. Amylase acts on α-1,4-glycosidic bonds of starch, cleav-
179
ing polysaccharides inside the chain and leading to formation of low-molecular hydrolysis
180
products, normal α-dextrins. Further hydrolysis gives maltose, maltotriose, and glucose. 39
181
Since the hydrolysis process results in biopolymer cleaving to smaller monomers, the viscos-
182
ity of the system decreases, and this phenomenon serves as a convenient analytical signal
183
to describe the catalytic process. Amylase is a thermally stable enzyme and has an opti-
184
mal temperature (Topt ) valued 70◦ C. To evaluate the catalytic activity of the composites,
185
amylase@ferra with 10 wt. % was selected and incubated with the starch solution at tem-
186
peratures varied from 60 to 90◦ C. For comparison, the process was performed using equal
187
amounts of free enzyme (Fig. 3). Evaluation of the process at 60◦ C demonstrated that the
188
amylase@ferria composite demonstrates catalytic activity and is able to hydrolyze the starch
189
solution. Compared to free amylase, the composite material showed approximately 1.5-fold
190
lower activity under these conditions, hydrolyzing starch in 34 minutes, while free enzyme
191
performed this process in 22 minutes (Fig. 3a). With elevation of the temperature to 70◦ C
192
accelerated the reaction rate of the both systems, but also makes the difference between free
193
enzyme and amylase@ferria even more pronounced: while free enzyme catalyzes the process
194
in 5 minutes, for the amylase@ferria composite the rate of the reaction was 2.5-folds lower
195
and required 13 minutes for hydrolysis of the same amount of the substrate (fig. 3b). The
196
difference between the reaction rates can be easily explained by the fact that in case of the
197
free amylase the reaction takes place in a homogeneous solution, while for the immobilized 9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
198
enzyme the system is heterogeneous and diffusion processes limits the reaction. While amy-
199
lase has an optimal temperature of 70◦ C, further elevation of the temperature to 80◦ C led to
200
its denaturation and inactivation processes. This is true for the free enzyme, and a notable
201
decrease in the reaction rate is observed, with prolongation of the hydrolysis time from 5
202
min at 70◦ C to 14 min at 80◦ C. In contrast, entrapped amylase showed the catalytic activity
203
almost identical to that at 70◦ C and close to the activity of the free enzyme (Fig. 3c). Fur-
204
ther increase of the reaction temperature to 90◦ C made the difference in the behavior of the
205
catalysts more obvious. While in the case of free amylase the starch hydrolysis was halted
206
due to the denaturation, the composite material demonstrated a high degree of catalytic
207
activity compared to the activity measured at 70 and 80◦ C (Fig. 3d). Preservation of the
208
catalytic activity by entrapped amylase at elevated temperatures is in good correlation with
209
our previous observations. 40 This phenomenon can be explained by tight interactions of the
210
enzyme’s tertiary structure with the inorganic matrix pore walls, resulting in stabilization
211
of the protein structure and its stabilization against harsh conditions.
212
One of the features of the amylase@ferria composite material is its magnetic responsive-
213
ness. The composite material can be separated from the reaction mixture by an external
214
magnetic field and be reused for another cycle of the catalytic process. Experiments showed
215
that the activity of the composite material remains essentially at the same level at least after
216
ten cycles of use (Fig. 3e), decreasing by only 4% in total and revealing a high stability of
217
the composite material and a prolonged profile of action, characteristic for the entrapped
218
enzymatic systems, when the activity is not related to the release of the enzyme. Another
219
important parameter of enzymatic formulations and composite materials is their storage sta-
220
bility. By their nature, enzymes are unstable in solutions and deteriorate over time. One of
221
the goals that can be achieved though enzyme immobilization is an increase of its stability
222
and prolongation of the storage period. 22,41 In order to evaluate the effect of entrapment
223
within sol-gel magnetite in amylase long-term stability the material was stored in phosphate
224
buffer solution (pH = 7.4) at 4◦ C for a period of 21 days and the activity was compared
10
ACS Paragon Plus Environment
Page 10 of 19
Page 11 of 19
Journal of Agricultural and Food Chemistry
225
to one measured for free enzyme (Fig. 3f). Entrapped amylase demonstrated significantly
226
higher stability at storage conditions compared to free enzyme. While free amylase lost all
227
its activity within 17 days, composite material demonstrates 91% of its initial activity after
228
21 days of storage with only 0.5%/day degradation rate. When compared to results reported
229
earlier it can be seen that immobilization of amylase by entrapment results in higher stability
230
compared to covalent bonding or adsorption. 22,42–45 This fact can be explained by tighter
231
three dimensional interaction of the enzyme with inorganic matrix and absence of covalent
232
bonds between the supporting material and the enzyme that can affect the conformation of
233
enzyme. 40
234
Amylase@magnetite ceramic catalytic coatings
235
As an alternative to application as a magnetically separable catalyst, amylase@magnetite
236
composite can be applied as a ceramic coating on a surface of a reactor. For this purpose,
237
mixture of amylase and stable magnetite hydrosol was applied on the vessel surface before
238
solvent removal by spraying, dip-coating or by using the Mayer rod. Upon condensation,
239
irreversible sol-gel transition occurred resulted in the formation of the enzymatically-doped
240
ceramic magnetite coating on the surface of the substrate. The thickness of the formed
241
ceramic layer depends on the amount of the applied colloid solution by the factor of 24,
242
(that is, application of the 5 micrometer layer of hydrosol mixture resulted in the formation
243
of the composite ceramic coating with the thickness of ≈210 nm) and can be precisely
244
regulated in that way. The formed coating showed a high degree of the homogeneity (Fig.
245
4a).
246
The composite bioceramic coating demonstrated high thermal stability similar to the
247
one observed on the magnetic particles (Fig. 4b). The relative activity of the composite
248
coating was stable at elevated temperatures without notable degradation at 70◦ C, Topt of
249
α-amylase, and only slight degradation of the material for 4% after incubation at 80◦ C after
250
90 min of incubation was observed. At 90◦ the enzymatic activity linearly decreased over 11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
251
the time, loosing 0.37% of its relative activity per minute, but still demonstrated higher
252
thermal stability than free enzyme, which was fully inactivated in 5 minutes at this tem-
253
perature. The performance of the model reactor was tested by evaluating the rate of starch
254
hydrolysis in successive catalytic cycles. For this purpose, the starch solution was subjected
255
to hydrolysis in batch experiments in the reactor at 80◦ C, the process was monitored by
256
measuring the viscosity of the solution every 5 minutes (Fig. 4c). When the viscosity of
257
solution reached the constant value, the starch solution was changed with a new portion and
258
the hydrolysis dynamic was measured again (for details see Experimental section). Viscosity
259
measurements in the set of experiments demonstrated the excellent stability of the model re-
260
actor performance in time. Starch solution hydrolysis demonstrated almost equivalent rates
261
in 10 successive experiments and took 20 minutes at 80◦ C with only minor degradation by
262
4%, which demonstrated the high stability of the hybrid amylase@ferria coating catalytic
263
properties. It can be concluded that sych and makes it promising material for application
264
in design of new types of advanced batch and flow reactors.
265
Acknowledgement
266
This work was supported by Russian Foundation for Basic Research, grant 18-33-01170
267
mol a. The authors declare no conflict of interests.
268
References
269
270
271
272
273
(1) Sanchez, C.; Belleville, P.; Popall, M.; Nicole, L. Chemical Society Reviews 2011, 40, 696–753. (2) Piccinno, F.; Gottschalk, F.; Seeger, S.; Nowack, B. Journal of Nanoparticle Research 2012, 14, 1109. (3) Lee, J.; Mahendra, S.; Alvarez, P. J. ACS nano 2010, 4, 3580–3590. 12
ACS Paragon Plus Environment
Page 12 of 19
Page 13 of 19
Journal of Agricultural and Food Chemistry
274
(4) Pansini, M.; Dell’Agli, G.; Marocco, A.; Netti, P. A.; Battista, E.; Lettera, V.; Ver-
275
gara, P.; Allia, P.; Bonelli, B.; Tiberto, P. Journal of Biomedical Nanotechnology 2017,
276
13, 337–348.
277
278
279
280
(5) Cushen, M.; Kerry, J.; Morris, M.; Cruz-Romero, M.; Cummins, E. Trends in Food Science & Technology 2012, 24, 30–46. (6) Rashidi, L.; Khosravi-Darani, K. Critical reviews in food science and nutrition 2011, 51, 723–730.
281
(7) Lin, Y.; Ren, J.; Qu, X. Accounts of chemical research 2014, 47, 1097–1105.
282
(8) Verma, M. L.; Barrow, C. J.; Puri, M. Applied microbiology and biotechnology 2013,
283
284
285
286
287
97, 23–39. (9) Wang, Z.-G.; Wan, L.-S.; Liu, Z.-M.; Huang, X.-J.; Xu, Z.-K. Journal of Molecular Catalysis B: Enzymatic 2009, 56, 189–195. (10) Jia, H.; Zhu, G.; Vugrinovich, B.; Kataphinan, W.; Reneker, D. H.; Wang, P. Biotechnology progress 2002, 18, 1027–1032.
288
(11) Ge, J.; Lu, D.; Liu, Z.; Liu, Z. Biochemical Engineering Journal 2009, 44, 53–59.
289
(12) Filippusson, H.; Hornby, W. Biochemical Journal 1970, 120, 215–219.
290
(13) Yoshimoto, M. Enzyme Stabilization and Immobilization: Methods and Protocols 2017,
291
292
293
9–18. (14) Liu, W.; Ye, A.; Singh, H. Microencapsulation and Microspheres for Food Applications. Sagis LMC, ed. Academic Press, New York, NY, USA. p 2015, 151–170.
294
(15) Koshel, E. I.; Chelushkin, P. S.; Melnikov, A. S.; Serdobintsev, P. Y.; Stolbovaia, A. Y.;
295
Saifitdinova, A. F.; Shcheslavskiy, V. I.; Chernyavskiy, O.; Gaginskaya, E. R.; Ko-
13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
296
shevoy, I. O. Journal of Photochemistry and Photobiology A: Chemistry 2017, 332,
297
122–130.
298
299
300
301
302
303
304
305
306
307
308
309
310
311
(16) Awasthi, M. K.; Pandey, A. K.; Bundela, P. S.; Wong, J. W.; Li, R.; Zhang, Z. Bioresource technology 2016, 213, 181–189. (17) Liang, K.; Coghlan, C. J.; Bell, S. G.; Doonan, C.; Falcaro, P. Chemical Communications 2016, 52, 473–476. (18) Shi, X.; Gu, W.; Li, B.; Chen, N.; Zhao, K.; Xian, Y. Microchimica Acta 2014, 181, 1–22. (19) Chapurina, Y. E.; Drozdov, A. S.; Popov, I.; Vinogradov, V. V.; Dudanov, I. P.; Vinogradov, V. V. Journal of Materials Chemistry B 2016, 4, 5921–5928. (20) Cui, J.; Feng, Y.; Yue, S.; Zhao, Y.; Li, L.; Liu, R.; Lin, T. Journal of Chemical Technology and Biotechnology 2016, 91, 1905–1913. (21) Li, J.; Jin, X.; Liu, Y.; Li, F.; Zhang, L.; Zhu, X.; Lu, Y. Chemical Communications 2015, 51, 9628–9631. (22) Kahraman, M. V.; Bayramo˘glu, G.; Kayaman-Apohan, N.; G¨ ung¨or, A. Food Chemistry 2007, 104, 1385–1392.
312
(23) Govan, J.; Gun’ko, Y. K. Nanomaterials 2014, 4, 222–241.
313
(24) Xu, J.; Sun, J.; Wang, Y.; Sheng, J.; Wang, F.; Sun, M. Molecules 2014, 19, 11465–
314
11486.
315
(25) Zhou, L.; Yuan, J.; Wei, Y. Journal of Materials Chemistry 2011, 21, 2823–2840.
316
(26) Dyal, A.; Loos, K.; Noto, M.; Chang, S. W.; Spagnoli, C.; Shafi, K. V.; Ulman, A.;
317
Cowman, M.; Gross, R. A. Journal of the American Chemical Society 2003, 125, 1684–
318
1685. 14
ACS Paragon Plus Environment
Page 14 of 19
Page 15 of 19
Journal of Agricultural and Food Chemistry
319
(27) Wu, Y.; Wang, Y.; Luo, G.; Dai, Y. Bioresource technology 2009, 100, 3459–3464.
320
(28) Safarik, I.; Baldikova, E.; Prochazkova, J.; Safarikova, M.; Pospiskova, K. 2018,
321
(29) Hosseini, S. H.; Hosseini, S. A.; Zohreh, N.; Yaghoubi, M.; Pourjavadi, A. Journal of
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
agricultural and food chemistry 2018, (30) Zahra, Z.; Waseem, N.; Zahra, R.; Lee, H.; Badshah, M. A.; Mehmood, A.; Choi, H.-K.; Arshad, M. Journal of agricultural and food chemistry 2017, 65, 5598–5606. (31) Shapovalova, O. E.; Drozdov, A. S.; Brushkova, E. A.; Morozov, M. I.; Vinogradov, V. V. Arabian Journal of Chemistry 2018, (32) Cornell, R. M.; Schwertmann, U. The iron oxides: structure, properties, reactions, occurrences and uses; John Wiley & Sons, 2003. (33) Li, Y.-S.; Church, J. S.; Woodhead, A. L. Journal of Magnetism and Magnetic Materials 2012, 324, 1543–1550. (34) Drozdov, A. S.; Ivanovski, V.; Avnir, D.; Vinogradov, V. V. Journal of colloid and interface science 2016, 468, 307–312. (35) Larson, S. B.; Greenwood, A.; Cascio, D.; Day, J.; McPherson, A. Journal of molecular biology 1994, 235, 1560–1584. (36) Shabanova, E. M.; Drozdov, A. S.; Ivanovski, V.; Suvorova, I. I.; Vinogradov, V. V. RSC Advances 2016, 6, 84354–84362. (37) Drozdov, A. S.; Vinogradov, V. V.; Dudanov, I. P.; Vinogradov, V. V. Scientific reports 2016, 6 . (38) Anastasova, E. I.; Ivanovski, V.; Fakhardo, A. F.; Lepeshkin, A. I.; Omar, S.; Drozdov, A. S.; Vinogradov, V. V. Soft matter 2017, 13, 8651–8660. (39) Layer, P.; Zinsmeister, A. R.; DiMagno, E. P. Gastroenterology 1986, 91, 41–48. 15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
342
343
(40) Drozdov, A. S.; Shapovalova, O. E.; Ivanovski, V.; Avnir, D.; Vinogradov, V. V. Chemistry of Materials 2016, 28, 2248–2253.
344
(41) Singh, V.; Singh, D. Process Biochemistry 2013, 48, 96–102.
345
(42) Sohrabi, N.; Rasouli, N.; Torkzadeh, M. Chemical Engineering Journal 2014, 240,
346
347
348
349
350
351
352
426–433. (43) Hasirci, N.; Aksoy, S.; Tumturk, H. Reactive and Functional Polymers 2006, 66, 1546– 1551. (44) Mukherjee, A. K.; Kumar, T. S.; Rai, S. K.; Roy, J. K. Biotechnology and Bioprocess Engineering 2010, 15, 984–992. (45) Ertan, F.; Yagar, H.; Balkan, B. Preparative biochemistry & biotechnology 2007, 37, 195–204.
16
ACS Paragon Plus Environment
Page 16 of 19
Page 17 of 19
Journal of Agricultural and Food Chemistry
Figure 3: Catalytic hydrolysis of starch solution by free amylase and amylase@ferria kinetic curves. Hydrolysis process kinetics at 60◦ C (a), 70◦ C (b), 80◦ C (c), and 90◦ C (d); relative activity of the amylase@ferria composite reused in ten successive experiments (e); long term stability of amylase@ferria and free amylase stored at 4◦ C in phosphate buffer solution (f). The results presented is represented by average values of 3 measurements for each of the curves.
17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 4: Enzymatically-doped ceramic coating amylase@ferria on the surface of the glass vessel. SEM cross section of the coating is presented (a); the dynamic of starch hydrolysis in the model reactor in successive cycles. Catalytic coating in model experiment have demonstrated a good stability against thermal inactivation and peeling, and showed high degree of reusability of the system with only 6% degradation after 10 cycles of starch hydrolysis.
18
ACS Paragon Plus Environment
Page 18 of 19
Page 19 of 19
Journal of Agricultural and Food Chemistry
199x268mm (300 x 300 DPI)
ACS Paragon Plus Environment