Macroporous silica microcapsules immobilizing esterase with high hydrolysis reactivity

Article abstract of DOI:10.1246/BCSJ.20200101

An esterase, 3,4-dihydrocoumarin hydrolase, was directly immobilized into silica microcapsules. The hydrolysis reaction of 3,4-dihydrocoumarin by a macroporous silica microcapsule immobilizing the esterase was faster than those by mesoporous ones. Using t

Full text of DOI:10.1246/BCSJ.20200101

Document type: Article
Table 1. Profiles of silica microcapsules
Short Article
Capsule
No.
Enzyme contenta
SSAb
PVc
PPDd
Precipitant
Macroporous Silica Microcapsules
Immobilizing Esterase
with High Hydrolysis Reactivity
(wt%)
(m2/g) (mL/g) (nm)
Cap0
Cap1
Cap2 (NH4)2SO4
Cap3 NH4HCO3
Cap4
Cap5e
Cap6f
Cap7g
NH4Cl
NH4Cl
®
449
273
377
293
263
333
292
446
0.343 3.71
0.258 3.28
0.413 4.19
0.412 4.19
0.536 9.23
0.362 4.19
0.381 4.76
0.419 4.76
18.7
14.4
15.7
11.3
11.4
15.7
2.5
KHCO3
KHCO3
KHCO3
KHCO3
Masahiro Fujiwara,*1 Shigeru Shoji,1
Yuka Murakami,1
and Kazuhiko Ishikawa2
aEstimated from the weight decrease from 200 to 500 °C in TG-
DTA analyses. bSpecific surface area calculated by BET plot.
cPore volume of mesopore estimated by BJH method using adsorp-
tion branches. dPeak pore diameter of mesopore estimated by BJH
method using adsorption branches. Prepared with 1 g of sodium
polyacrylate. fPrepared with 2 g of the polymer. gPrepared with 3 g
of the polymer.
1National Institute of Advanced Industrial
M. Fujiwara
Science and Technology (RICPT; Tohoku Center), 4-2-1
Nigatake, Miyagino-ku, Sendai, Miyagi 983-8551, Japan
e
2National Institute of Advanced Industrial Science
and Technology (BRI, Kansai Center),
1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan
OH
O
O
Esterase
+ H2O
E-mail: m-fujiwara@aist.go.jp
Received: April 2, 2020; Accepted: May 7, 2020;
Web Released: May 14, 2020
COOH
3-(2-Hydroxyphenyl)propionic acid
3,4-Dihydrocoumarin
Scheme 1. Hydrolysis reaction of 3,4-dihydrocoumarin.
Abstract
Acinetobacter calcoaceticus F46, because this esterase is reported
to catalyze the hydrolysis of 3,4-dihydrocoumarin very rapidly,15
being beneficial for evaluating the effects of the pore properties
of silica microcapsules on the reaction rate of the enzyme. In this
article, we report the behaviors of the hydrolysis reactions of car-
boxylic acid esters catalyzed by the esterase immobilized into silica
microcapsules toward developing our microcapsule technology.
The esterase was immobilized into silica microcapsules using
silica precipitants such as NH4Cl, (NH4)2SO4, NH4HCO3 and
KHCO3.9 A W/O (water/oil) emulsion, where the water phase was
a sodium silicate solution with the esterase and the oil phase was n-
hexane solution with Tween 85, was added to silica precipitant
solutions to form W/O/W emulsion. Silica particles formed in the
resulting solutions encapsulated the esterase directly (see support-
ing information). The profiles of microcapsules thus prepared are
summarized in Table 1. While silica nanoparticles smaller than
100 nm may possibly be toxic to living organisms,16,17 the parti-
cle sizes of all microcapsules were more than 100 nm as shown
in Figure S4. It is thought that these silica microcapsules are
ascertained to be safe materials. The hydrolysis reactions of 3,4-
dihydrocoumarin by the esterase were estimated by monitoring the
absorption intensities of the hydrolyzed product, 3-(2-hydroxy-
phenyl)propionic acid, at the wavelength of 270 nm (Scheme 1).15
The time variations of the absorption of reaction solutions with
various silica microcapsules are illustrated in Figure 1. When 0.3
mg of the original esterase (non-immobilized) was used, the
absorption at wavelength of 270 nm was approximately stable at
around intensity 1.5 (not shown in Figure 1). This meant that the
hydrolysis reaction was immediately completed before the spec-
trophotometric measurement was started (within 1 min).
An esterase, 3,4-dihydrocoumarin hydrolase, was directly
immobilized into silica microcapsules. The hydrolysis reaction
of 3,4-dihydrocoumarin by a macroporous silica microcapsule
immobilizing the esterase was faster than those by mesoporous
ones. Using this macroporous microcapsule, the hydrolysis reac-
tion of p-nitrophenyl acetate proceeded with comparable rate to
non-immobilized esterase.
Keywords: Macroporous
j
Silica microcapsule
j
Enzyme immobilization
Immobilization of enzymes is an essential technology for their
practical and industrial applications.1-4 Since amorphous silicas are
generally safe materials (for example; used as food additives) and
practically insoluble in common water and organic solvents, they
are well used as the supports of immobilized enzymes.5-8 However,
their small pore voids often restrict the access of reactants to the
immobilized enzymes to slow reaction rates. We have studied the
preparations of amorphous silica microcapsules using W/O/W
(water/oil/water) emulsion with sodium silicate solution.9-14 When
enzymes are added to the solution, the enzymes are directly immo-
bilized into the silica microcapsules.12-14 Hyperthermophilic β-
glucosidase immobilized into the microcapsule is effective for the
hydrolysis reaction of cellobiose.14 However, the reaction rate is
significantly slower than that of non-immobilized one, because the
narrow mesopore of the silica microcapsule (about 2.6 nm) sup-
presses the access of cellobiose to β-glucosidase. Therefore, the
optimization of the pore properties of the microcapsule is required.
On the other hand, we also found methods for producing silica
microcapsules bearing macropores by the addition of water-soluble
polymers to a sodium silicate solution.10,11 It is expected that
macroporous silica microcapsule encapsulating enzyme facilitates
the access of reactant to enhance the reaction rate. Here we focus on
the preparation of silica microcapsules immobilizing an esterase,
3,4-dihydrocoumarin hydrolase [EC 3.1.1.35] obtained from
For balancing the amount of the esterase (its maximum content
was up to 20 wt% in microcapsules as listed in Table 1), 1.5 mg of
silica microcapsules immobilizing the esterase (Cap1-Cap4) were
employed for the hydrolysis reaction. As shown in Figure 1, when
a vacant microcapsule without the esterase (Cap0) was used (1.5
Bull. Chem. Soc. Jpn. 2020, 93, 1043–1045 | doi:10.1246/bcsj.20200101
© 2020 The Chemical Society of Japan | 1043

(A)
(B)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
Cap4
Cap3
Cap1
Cap0
Cap2
(C)
(D)
0
5
10
15
20
25
30
Reaction time (min)
Figure 1. Time variation of the absorption intensity at wave-
length of 270 nm in the hydrolysis reaction of 3,4-dihydrocou-
marin using silica microcapsules immobilizing the esterase.
mg), the increase of the absorption intensity at 270 nm was slight
even after 30 min. The vacant silica microcapsule was nearly inert
for this reaction. On the other hand, the intensity elevations of the
absorption were clearly observed when the microcapsules encap-
sulating the esterase were used (Cap1-Cap4). Thus, the hydrolysis
reaction was catalyzed by the esterase included in the micro-
capsules. The intensity elevations at the early stage accurately
displayed the enzymatic activities of these microcapsules. Rapid
elevations of the intensity were found in the reactions using the
microcapsules prepared with NH4HCO3 (Cap3) and KHCO3
(Cap4). These reactions nearly completed within 5 min, because
no further increases of the absorption intensity were observed. It
appears that Cap4 with larger pore volume and peak mesopore
diameter than Cap3 was superior to Cap3. On the other hand, the
reactions using microcapsules prepared with NH4Cl (Cap1) and
(NH4)2SO4 (Cap2) were slower. Since Cap1 with the highest
content of the esterase (18.7 wt%) showed the lowest reaction rate,
the enzymatic activities of these microcapsules had no relation to
the contents of the esterase. The difference of the activity between
Cap2 and Cap3 that had the same pore volume is caused from the
different distribution of mesopores shown in Figure S2. Higher
volume of the mesopores of Cap3 around 4 nm sufficiently larger
than 3,4-dihydrocoumarin resulted in faster reaction rate. It is
thought that the reaction rates were significantly influenced by the
pore structures of silica microcapsules, because more porous Cap4
had the highest activity. On the other hand, the esterase encap-
sulated into microcapsule was comparatively thermally stable,
because the enzymatic activity was nearly unchanged when Cap3
treated at 50 °C was used. However, the treatment at 80 °C moder-
ately reduced the activity as indicated in Figure S5.
We reported before that the addition of suitable water-soluble
polymers into a sodium silicate solution of W/O/Wemulsion prod-
uces silica microcapsules bearing macropores.10 Thus, this tech-
nique was applied to the immobilization of the esterase. In this
study, sodium polyacrylate (Mw: ³2100) was used because of its
high solubility to water. KHCO3 was employed as precipitant in this
preparation. From 1 to 3 g of the polymer was added to the solution
of 29.8 g of sodium silicate, 4 g of the esterase and 13 mL of dis-
tilled water (Cap5-Cap7). As listed in Table 1, both the volumes
and peak diameters of mesopore of these microcapsules (Cap5-
Cap7) were lower than those of Cap4. Our previous paper10
reported that mesopores of the microcapsules were expanded to
macropores by the addition of water-soluble polymers, resulting in
the decrease of their mesoporosity. In SEM images of Figure 2A,
no pores observable by common SEM analysis were found in the
Figure 2. Scanning microscope images of silica microcapsules.
(A) Cap4, (B) Cap5, (C) Cap6 and (D) Cap7.
1.6
Cap6
1.4
Cap4
1.2
Cap5
1.0
0.8
0.6
0.4
Cap7
0
5
10
15
20
25
30
Reaction time (min)
Figure 3. Time variation of the absorption intensity at wave-
length of 270 nm in the hydrolysis reaction of 3,4-dihydro-
coumarin using silica microcapsules encapsulating the esterase
prepared with sodium polyacrylate.
microcapsules prepared without the polymer (Cap4). In the micro-
capsule Cap5 prepared with 1 g of the polymer, cavities inside the
microcapsule were observed as darkish spots (Figure 2B). On the
other hand, in the microcapsules obtained using 2 g of the polymer
(Cap6), macropores in the range of 100 to 500 nm were clearly
observed (Figure 2C). Many cavities as darkish spots were also
found. In the microcapsules obtained with 3 g of the polymer
(Cap7), macropores larger than 1 ¯m were clearly observed
(Figure 2D). Thus, the addition of the polymer fabricated macro-
pores in microcapsules.
Figure 3 illustrates the reaction rate of the hydrolysis reaction
of 3,4-dihydrocoumarin catalyzed by these silica microcapsules
immobilizing the esterase. Although the microcapsule prepared
with 1 g of the polymer (Cap5) showed a comparable reaction rate
to Cap4 that was obtained without the polymer, the macroporous
microcapsule prepared with 2 g of the polymer (Cap6) displayed
the highest reaction rate. The intensity elevation of the absorption
at the wavelength of 270 nm was very rapid and the reaction was
completed within 2 min. It is thought that the macropores in the
microcapsule Cap6 substantially facilitated the access of the reac-
tant to the esterase, achieving this fast reaction. On the other hand,
when the microcapsule obtained with 3 g of the polymer (Cap7)
was used, the reaction was slow and unfinished even after 30 min.
1044 | Bull. Chem. Soc. Jpn. 2020, 93, 1043–1045 | doi:10.1246/bcsj.20200101
© 2020 The Chemical Society of Japan

100
80
60
40
20
0
esterase (diffusion-controlled reaction) but by the reactivity of the
esterase (reaction-controlled reaction).
In conclusion, an esterase obtained from Acinetobacter calcoa-
ceticus F46 was successfully immobilized into silica microcap-
sules. The esterase encapsulated in the silica microcapsules had
hydrolysis activity toward esters. The reaction rate of the hydroly-
sis of 3,4-dihydrocoumarin by the esterase was mainly influenced
by the pore properties of the silica microcapsules. A silica micro-
capsule that had macropores up to 500 nm showed the fastest
reaction rate. The wide macropore facilitating the access of the
reactant to the esterase promoted the reaction. This macroporous
silica microcapsule nearly had an enzymatic activity equivalent
to the non-immobilized esterase in the hydrolysis reaction of p-
nitrophenyl acetate. The preparation technique of macroporous
silica microcapsules will contribute to the advancement of the
technology of enzyme immobilization.
non-immobilized
Cap6
0
1
2
3
4
5
6
Reaction time (h)
Figure 4. Time variations of the yield of p-nitrophenol from p-
nitrophenyl acetate catalyzed by the non-immobilized esterase
and the silica microcapsule immobilizing the esterase Cap6
(per 5 mg of the esterase).
The silica microcapsule immobilizing the esterase bearing macro-
pores from 100 to 500 nm (Cap6) had an excellent activity for the
hydrolysis reaction.
This study was supported by JST Regional Industry-Academia
Value Program of 2017 FY (VP29117938869). Authors thank Dr.
Naoki Mimura (RICPT, AIST) for his help in the experiments.
As listed in Table 1, the content of the esterase in Cap7 was
estimated to be about 2.5 wt%, while all other microcapsules
included more than 10 wt% of the esterase by TG-DTA analysis.
The low activity of Cap7 toward the hydrolysis of 3,4-dihydro-
coumarin resulted from poor content of the esterase. It is likely that
the macropore larger than 1 ¯m in Cap7 is too large to encapsulate
the esterase, and a great portion of the esterase was eliminated
during the preparation process. On the other hand, since the size
of macropores in Cap6 was appropriate, this silica microcapsule
contained sufficient amount of the esterase to show the highest
performance in the hydrolysis reaction. The molecular size of the
esterase (molecular mass: 60 kDa)15 is likely to be comparable to
that of bovine serum albumin (molecular mass: 66.4 kDa; mo-
lecular dimension 5.0 © 7.0 © 7.0 nm3),13 which is greatly smaller
than the macropore of Cap6. The recycling of Cap6 was exam-
ined as illustrated in Figure S6. Although the enzymatic activity
gradually decreased with recycling, the hydrolysis reaction was
completed after about 10 min even in the tenth reaction (ninth
recycling). Despite Cap6 having enormously larger macropores
than the esterase, the leaching of esterase by recycling scarcely
occurred and it is recyclable for the reaction.
Although the reaction rate of the hydrolysis of 3,4-dihydrocou-
marin by the macroporous silica microcapsule Cap6 was definitely
quick, the rate by non-immobilized esterase is much faster because
this enzyme is specific to the reaction. Then, we attempted another
reaction, the hydrolysis of p-nitrophenyl acetate, which is well
examined for the evaluation of esterase enzymes.18 The reaction
rates of this hydrolysis were compared between the immobilized
and non-immobilized esterase. 5 mg of the non-immobilized
esterase and 30 mg of the silica microcapsule encapsulating the
esterase Cap6 were used for balancing the esterase contents
between them. In this case, the reaction was monitored by the
intensity of the absorption of p-nitrophenol as hydrolyzed product
at the wavelength of 400 nm. As illustrated in Figure 4, the time
variation of the yield of p-nitrophenol in the reaction with Cap6
was closely overlapped with that of the non-immobilized esterase.
The esterase immobilized in the macroporous silica microcapsule
had nearly the same activity as the non-immobilized esterase for
the hydrolysis reaction of p-nitrophenyl acetate, because the large
macropore of the silica microcapsule Cap6 scarcely reduced the
reaction rate by the esterase. In this reaction, it is thought that the
reaction was controlled not by the access of the reactant to the
Supporting Information
Experimental procedures, nitrogen sorption isotherms, the BJH
mesopore distribution, SEM images, and particle size distribution
of silica microcapsules. This material is available on https://
doi.org/10.1246/bcsj.20200101.
References
1
M. Komiyama, T. Mori, K. Ariga, Bull. Chem. Soc. Jpn. 2018,
91, 1075.
2
F.-K. Shieh, S.-C. Wang, C.-I. Yen, C.-C. Wu, S. Dutta, L.-Y.
Chou, J. V. Morabito, P. Hu, M.-H. Hsu, K. C.-W. Wu, C.-K. Tsung,
J. Am. Chem. Soc. 2015, 137, 4276.
3
4
5
R. A. Sheldon, S. van Pelt, Chem. Soc. Rev. 2013, 42, 6223.
N. Song, Y.-W. Yang, Chem. Soc. Rev. 2015, 44, 3474.
J. Livage, T. Coradin, C. Roux, J. Phys.: Condens. Matter
2001, 13, R673.
6
7
E. Magner, Chem. Soc. Rev. 2013, 42, 6213.
Y. Bao, C. Shi, T. Wang, X. Li, J. Ma, Microporous
Mesoporous Mater. 2016, 227, 121.
J. Sun, D. Zhang, W. Zhao, Q. Ji, K. Ariga, Bull. Chem. Soc.
Jpn. 2019, 92, 275.
8
9
M. Fujiwara, K. Shiokawa, Y. Tanaka, Y. Nakahara, Chem.
Mater. 2004, 16, 5420.
10 M. Fujiwara, K. Shiokawa, I. Sakakura, Y. Nakahara, Nano
Lett. 2006, 6, 2925.
11 M. Fujiwara, K. Shiokawa, I. Sakakura, Y. Nakahara, Lang-
muir 2010, 26, 6561.
12 M. Fujiwara, K. Shiokawa, K. Hayashi, K. Morigaki, Y.
Nakahara, J. Biomed. Mater. Res., Part A 2007, 81A, 103.
13 M. Fujiwara, K. Shiokawa, K. Morigaki, Y. Zhu, Y. Nakahara,
Chem. Eng. J. 2008, 137, 14.
14 M. Watanabe, M. Fujiwara, K. Ishikawa, Chem. Lett. 2014, 43,
1272.
15 M. Kataoka, K. Honda, S. Shimizu, Eur. J. Biochem. 2000,
267, 3.
16 Y. Li, L. Sun, M. Jin, Z. Du, X. Liu, C. Guo, Y. Li, P. Huang, Z.
Sun, Toxicol. In Vitro 2011, 25, 1343.
17 D. Napierska, L. C. J. Thomassen, V. Rabolli, D. Lison, L.
Gonzalez, M. Kirsch-Volders, J. A. Martens, P. H. Hoet, Small 2009, 5,
846.
18 W. N. Aldridge, Biochem. J. 1953, 53, 110.
Bull. Chem. Soc. Jpn. 2020, 93, 1043–1045 | doi:10.1246/bcsj.20200101
© 2020 The Chemical Society of Japan | 1045