Enzyme-Decorated Covalent Organic Frameworks as Nanoporous Platforms for Heterogeneous Biocatalysis

Article abstract of DOI:10.1002/chem.201903807

Sustainability in chemistry heavily relies on heterogeneous catalysis. Enzymes, the main catalyst for biochemical reactions in nature, are an elegant choice to catalyze reactions due to their high activity and selectivity, although they usually suffer from lack of robustness. To overcome this drawback, enzyme-decorated nanoporous heterogeneous catalysts were developed. Three different approaches for Candida antarctica lipase B (CAL-B) immobilization on a covalent organic framework (PPF-2) were employed: physical adsorption on the surface, covalent attachment of the enzyme in functional groups on the surface and covalent attachment into a linker added post-synthesis. The influence of the immobilization strategy on the enzyme uptake, specific activity, thermal stability, and the possibility of its use through multiple cycles was explored. High specific activities were observed for PPF-2-supported CAL-B in the esterification of oleic acid with ethanol, ranging from 58 to 283 U mg^?1, which was 2.6 to 12.7 times greater than the observed for the commercial Novozyme 435.

Full text of DOI:10.1002/chem.201903807

A Journal of
Accepted Article
Title: Enzyme-Decorated Covalent Organic Framework as a
Nanoporous Platform for Heterogeneous Biocatalysis
Authors: Felipe L. Oliveira, Stefania P. de Souza, Jonathan Bassut,
Heiddy M. Álvarez, Yunier Garcia-Basabe, Rodrigo O. M.
Alves de Souza, Pierre M. Esteves, and Raoni Schroeder
Borges Goncalves
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201903807
Link to VoR: http://dx.doi.org/10.1002/chem.201903807
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10.1002/chem.201903807
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Enzyme-Decorated Covalent Organic Framework as a
Nanoporous Platform for Heterogeneous Biocatalysis
Felipe L. Oliveira,[a] Stefania P. de Souza,[b] Jonathan Bassut,[b] Heiddy M. Álvarez,[c] Yunier Garcia-
Basabe,[a] Rodrigo O. M. Alves de Souza,[b] Pierre M. Esteves,*[a] Raoni S. B. Gonçalves*[a]
[10,11]
Porous materials allow the storage of gases,
gas capture
and can be
Abstract: Sustainability on chemistry heavily relies on heterogeneous
catalysis. Enzymes, the main catalyst for biochemical reactions in
nature, are an elegant choice to catalyze reactions due to their high
activity and selectivity, although they usually suffer from lack of
robustness. To overcome this drawback, enzyme-decorated
nanoporous heterogeneous catalysts were developed. Three different
approaches for Candida antarctica lipase B (CAL-B) immobilization
into a covalent organic framework (PPF-2) were employed: physical
adsorption on the surface, covalent attachment of the enzyme in
functional groups on the surface and covalent attachment into a linker
added post-synthesis. The influence of the immobilization strategy on
the enzyme uptake, specific activity, thermal stability, and the
possibility of its use through multiple cycles was explored. High
specific activities were observed for PPF-2 supported CAL-B in the
esterification of oleic acid with ethanol, ranging from 58 to 283 U/mg,
which was 2.6 to 12.7 times greater than the observed for the
commercial Novozyme 435.
[12]
[13]
[14]
and separation,
energy storage,
catalysis,
used in the development of electronic devices.[15] However, it is in
the development of new catalysts that these substances could
display one of their most prominent application. After the
unquestionable success of zeolites in catalytic chemistry, the next
step started with the development of new hybrid and fully organic
porous materials. These materials provide the opportunity to
manipulate their building blocks from the atomic level, allowing
the construction of complex architectures with the presence of
specific functional groups. This characteristic became a powerful
tool for the rational design of new catalysts.[3,16]
On the other hand, enantiomerically pure or enantio-enriched
compounds are fundamental targets for the synthesis of a wide
range of chemical products and their preparation displays a
fundamental role in several areas of the chemical industry, such
as pharmaceutical, agrochemical, fragrances, and food
additives.[17–19] However production of these compounds remains
a challenge [20,21] and, despite the enormous advances observed
in the last years, there is still a need for the development of simple
and scalable methods to produce such compounds with high
purity in an environmentally friendly manner.
Introduction
Enzymes are a smart and elegant option to catalyze reactions due
to their high activity and selectivity. Among different strategies to
produce optically active compounds, a promising alternative is the
use of lipases (triacylglycerol hydrolases E.C. 3.1.1.3), such as
Candida antarctica Lipase B (CAL-B), for kinetic resolution
reactions.[22],[23] Due to their capacity to perform reactions under
mild conditions, the possibility of their use in organic solvents,
broad substrate specificity, and high stereoselectivity, these
biocatalysts have been widely used for the resolution of alcohols,
amines, lactones, and carboxylic acids.[24–26] However, some
issues such as the limited range of temperature, recyclability, and
long-term stability stand as challenges for the utilization of these
biocatalysts in large scale production.[27,28] A widely used strategy
to overcome these problems is the immobilization of the enzyme
into a solid substrate.[29,30] Reported for the first time in 1916,[31]
this method has grown significantly over the past decades
allowing the economic viability of the use of several enzymes in
industrial processes causing a great economic and scientific
impact. The immobilization of enzymes in a heterogeneous
substrate promotes a convenient handling and a facile separation
of the biocatalyst from the reaction medium.[32] Additionally, it
often enhances enzyme stability towards denaturation by
autolysis, decomposition by organic solvents or by heat, as well
as boosting the enzyme activity. [33]
During the last years, the understanding, design, and
manipulation of nanoporous materials emerged as a fundamental
area in modern chemistry, displaying a key role in the frontier for
innovative technologies.[1,2] Especially, organic nanoporous
structures have been gaining singular attention along the last
[1,3,4]
decade, including covalent organic framework (COFs)
and
related structures. [5–9]
[a]
F. L. Oliveira, Dr. Y.G. Basabe, Prof. P.M. Esteves, Prof. R.S.B.
Gonçalves
Instituto de Química
Universidade Federal do Rio de Janeiro
Av. Athos da Silveira Ramos, 149 - Cidade Universitária, Rio de
Janeiro – RJ, 21941-909 - Brazil
E-mail: pesteves@iq.ufrj.br, raoni.schroeder@iq.ufrj.br
[b]
[b]
Dr. S. P. de Souza, Dr. J. Bassut, Prof. R.O.M.A de Souza
Biocatalysis and Organic Synthesis Group, Chemistry Institute
Universidade Federal do Rio de Janeiro
Av. Athos da Silveira Ramos, 149 - Cidade Universitária, Rio de
Janeiro – RJ, 21941-909 - Brazil
Profa. H. M. Álvarez
Departamento de Ciências Exatas
Universidade Estadual de Feira de Santana
Av. Transnordestina, S/N. Novo Horizonte, 252, Feira de Santana.
BA, 44036-900 - Brazil
There are several materials that are suitable for enzyme
immobilization, yet one of the most commercially successful
catalysts is Novozyme® 435 (CAL-B immobilized in Lewatit VP OC
1600 poly (methyl methacrylate-co-divinylbenzene)). Among all
the possible materials for enzyme immobilization, porous
structures like porous silica,[34,35] zeolites and metal-organic
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201903807
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frameworks (MOFs)[36–38] have been gaining popularity due to
their unique characteristics, notably their high surface area and
porosity. However, despite these notable advantages, some
issues like the absence of specific interactions with the enzymes,
problematic water stability, and their inorganic structure open
space for the development of new and better supports for
immobilization. Recently covalent organic frameworks (COFs)
have been reported as a host matrix for enzyme immobilization
COF synthesis
The COFs based on PPF-2 structure were synthesized as
previously described by Zhu and coworkers,[40] with slight
modifications. Briefly, PPF-2 was obtained through the reaction
between
methane) 0.788 mmol and
3
equivalents of TTA (tetrakis-(4-aminophenyl)-
equivalents of TFP (1,3,5-
4
triformylphloroglucinol) 1.05 mmol, under solvothermal conditions,
using 1,4-dioxane as the solvent, in the presence of 3M acetic
acid as catalyst. To create PPF-2 variations with the presence of
free amino or aldehyde groups on the structure surface, PPF-2-
NH2 and PPF-2-CHO were synthesized with a large excess of
TTA (3 equivalents of TTA 1.0 mmol and 2 equivalents of TFP
0.667 mmol) or a large excess of TFP (3 equivalents of TTA 0.562
mmol and 8 equivalents of TFP 1.5 mmol), respectively. The
PPFs were washed with 1,4-dioxane followed by THF
(tetrahydrofuran) and soaked on THF for 3 days to remove
unreacted monomers from the pores. The mass yields were 88%
for PPF-2, 88% for PPF-2-CHO and 65% for PPF-2-NH2.
that can present an enhancement of enzyme performance and
[39]
stability,
showing that porous organic materials could play a
major role as support for enzyme immobilization. Some
advantages of using COFs are the more precise control of their
chemical structure through the use of specific building blocks and
the enhanced stability in aqueous media as well in organic
solvents when compared with MOFs. Typical building blocks used
in COF synthesis are linked by robust covalent bonds and the
frameworks are stable to temperatures exceeding 300°C.
Specifically, in the use as enzyme support, the improved
versatility of COF’s building blocks allows the design of
frameworks with improved affinity with specific enzymes and also
the development of new strategies for enzyme immobilization.
The surface functionalization with epichlorohydrin was achieved
reacting PPF-2-NH2 with epichlorohydrin in 1,4-dioxane as
solvent under heating at 40°C and stirring for 2h. Throughout the
article, every reference to PPF-2-NH2 actually refers to the
functionalized variation with epichlorohydrin, since in the non-
functionalized variant there was no significant immobilization of
CAL-B. Figure 2-a shows a schematic representation of the
synthetic and functionalization approach.
In the present contribution, we explore an imine-linked covalent
organic framework, named PPF-2,[40] as a platform for CAL-B
immobilization. By tuning the functional groups on COFs’ outer
surface, both by synthesis or post-synthesis, we evaluate three
different strategies for enzyme immobilization: physical
adsorption, covalent attachment on the surface and covalent
attachment on a linker added post-synthesis on the surface
(Figure 1). The influence of the immobilization approach on the
enzyme uptake, specific activity, and thermal stability was
evaluated.
The COFs were characterized by FT-IR, 13C CPMAS NMR, and
N2 sorption measurements. As previously reported, PPF-2 exists
as the keto-enamine form instead of the enol-imine. Infrared
spectroscopy of PPF-2, PPF-2-NH2, and PPF-2-CHO exhibited
absorption bands at 1616 cm-1 for the C=O stretching, 1578 cm-1
for the C=C stretching, 1450-1500 cm-1 for aromatic ring
stretching, and 1225-1280 cm-1 for C-N stretching (Figure S24),
also confirming the keto-enamine COF structure.
Covalent attachment
by N-terminal
Covalent attachment
by epoxide group
Physical
adsorption
The N2 sorption measurements show that all the three structures
presented permanent porosity with
a
Type-I isotherm,
characteristic of microporous materials, with mean pore size
about 11 Å based on the Nonlocal Density Functional Theory
(NLDFT), which characterizes them as microporous according to
IUPAC.[41] PPF-2 presented a BET apparent specific surface area
of 1603 m2/g (Figure 2-b), slightly higher than the reported by Zhu
and coworkers (1470 m2/g). However, a decrease in the apparent
surface area was observed for PPF-2-CHO (Figure 2-c) and
PPF-2- NH2 (Figure 2-d), presenting 562 m²/g and 522 m²/g,
respectively.
A conceivable explanation for this decrease on the surface area
is that the building-block excess could lead to the formation of
small oligomers that would be trapped inside the structure,
thereby limiting the number of accessible pores, and decreasing
the specific surface area. In fact, there was no meaningful change
in pore size distribution, indicating that there was no structural
change due to variation in the building blocks ratio. Since enzyme
immobilization is expected to occur on the external surface of the
material, and the BET surface area is dictated by the internal area
due to the microporous structure the observed reduction may not
cause a profound influence on overall performance.
Figure 1. Schematic representation of the enzyme immobilization on COFs.
Tree different immobilization strategies could be explored based on the
chemical composition of the COF surface.
Results and Discussion
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Covalent attachment
by N-terminal
a)
Free -CHO group
Free –NH2 group
AcOH (cat.)
1,4-dioxane
120°C, 72h
1 ml CalB
+
phosphate buffer, pH 7
40°C, 40h
epichlorohydrin
1,4-dioxane
40°C, 4h
Physical
adsorption
Covalent attachment by
the epoxide group
Free epoxide group
PPF-2-NH2
PPF-2
PPF-2-CHO
200
c)
200
175
150
125
100
75
Adsorption
Desorption
b)600
500
400
300
200
100
0
Adsorption
Desorption
d)
Adsorption
Desorption
175
150
125
100
75
Pore Size Distribution
Pore Size Distribution
Pore Size Distribution
0.08
0.06
0.04
0.02
0.00
11.2 Å
11.2 Å
11.8 Å
0,04
0,04
0,02
0,02
0,00
50
50
25
25
0,00
0.4
5
10
15
20
25
30
5
10
15
20
25
30
5
10
15
20
25
30
Pore Size (Å)
Pore Size (Å)
Pore Size (Å)
0
0.0
0
0.0
0.0
0.2
0.4
0.6
0.8
1.0
0.2
0.4
0.6 0.8
1.0
0.2
0.6
0.8
1.0
Relative Pressure (P/Po)
Relative Pressure (P/Po)
Relative Pressure (P/Po)
Figure 2. a) Schematic representation of PPF-2 synthesis, functionalization, and enzyme immobilization. For simplicity, the COF structure was represented in the
enol-imine form. b-d) N2 adsorption-desorption isotherms at 77K for COFs and NLDFT pore size distribution histograms (inserts).
The chemical composition of the outer surface of the COFs was
characterized by X-ray photoelectron spectroscopy (XPS). The
core-level spectra of C1s and O1s for the materials before and
after enzyme immobilization are shown in Figure 3, and the
relative atomic concentrations are displayed in Table 1. The C1s
spectra of PPF-2, PPF-2-CHO, and PPF-2-NH2 (Figures 3a, 3b,
and 3c respectively) can be deconvoluted into five components.
The peaks at 284.6 eV in the three spectra can be attributed to C-
C and C-H. Carbon bonded to nitrogen (C–N) and hydroxyl
groups (C–OH) are located at 285.6, 285.5 and 285.9 eV, for
PPF-2, PPF-2-CHO and PPF-2-NH2, respectively. Peaks at 286.2,
286.5, and 286.6 eV were attributed to C=N. Carbon atoms in the
C=O group were observed at 288.1, 288.7 and 289.2 eV and the
peaks at 291.5, 290.7 and 290.9 eV were attributed to the C(H)=O
of the aldehyde group. Signals referent to C=O, C-O, and O-H
were also observed in the spectra of O1s (Figures 3d, 3e, and
3f). [42–44]
After enzyme immobilization, intense additional peaks were
observed at 288.2 eV for CAL-B@PPF-2, 287.8 eV for CAL-
B@PPF-2-CHO and 288.3 eV for CAL-B@PPF-2-NH2 (Figures
3a, 3b and 3c). These peaks were assigned to the C=O groups
of peptide bonds[45], as expected since the enzyme is deposited
on the surface of the materials.
Enzyme immobilization
Conjugation of PPF-2 was performed through physical adsorption
of CAL-B on PPF’s surface. For PPF-2-NH2, the activation of the
free amino groups was achieved by reaction of the material with
epichlorohydrin. Then, the activated material was treated with
CAL-B to afford the covalent conjugation with the free epoxy
group. Regarding to PPF-2-CHO, we postulated that the free
aldehyde groups could react with an available amino group in the
N-terminal residue of the protein and a covalent conjugation could
be performed through an imine bond formation, without previous
activation.
A nitrogen-oxygen ratio (N/O) of 0.72 was observed for the PPF-
2. Since both PPF-2-CHO and PPF-2- NH2 were synthesized with
excess of one type of the building blocks, it is expected that this
approach will generate a change in the surface composition, thus
increasing the N/O ratio for PPF-2-NH2 and decreasing for PPF-
Since CAL-B is larger (30 Å x 40 Å x 50 Å [46]) than the average
pore size of PPFs (11 Å), it is reasonable to state that only
superficial attachment of the enzyme is possible. The surface
functionalization, enzyme immobilization, and protein loading
determination protocol are described in detail in the Experimental
section.
2-CHO. As expected, PPF-2-NH2 presented
a
greater
nitrogen/oxygen ratio than PPF-2 (0.94), consequence of free
NH2 groups on the surface due to the excess of TTA during the
synthesis, while PPF-2-CHO presented a smaller N/O ratio (0.35),
due to the free CHO groups on the surface showing that the
surface composition can be modulated by changing the molar
ratio between the building blocks in the synthesis.
The surface composition analysis by XPS shows a substantial
change after the enzyme immobilization, displayed in Table 1,
and in all the three cases the N/O ratio changed, converging to a
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10.1002/chem.201903807
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value close to 0.65, therefore demonstrating that the enzyme
immobilization occurred successfully on the surface of the
materials. In fact, Figure 4 and Table 2 show that does not occur
a meaningful change in BET surface area and pore volume for
PPF-2-CHO and PPF-2-NH2. Since the greatest contribution to
the surface area is due to micropores, which are smaller than
CAL-B, it is quite reasonable to conclude that only the external
surface of the material was altered by the enzyme immobilization.
On the other hand, surprisingly PPF-2 showed a substantial
reduction in surface area and pore volume. Since the internal
region of the pores presents a nonpolar environment, denatured
enzymes could enter and absorb into the pores. This could
reduce the accessibility of the internal region of the material and,
consequently, decreases its apparent surface area and pore
volume. This also may decrease the enzymatic activity in this
catalyst when compared to its equivalents, since denatured
enzymes inside of the pores are not active. This fact will be
addressed later.
determined as 3.1±0.5 mg g-1 for PPF-2, 3.9±0.2 mg g-1 for PPF-
2-NH2 and 1.2±0.5 mg g-1 for PPF-2-CHO.
It is described that porous supports with higher surface areas
usually allow a higher enzyme loading [47]. However, comparisons
of enzyme loading and specific surface area of the COFs show
that, in this case, there are no correlations between surface area
and enzyme loading. Considering that the enzyme could not fit
into the pores due to its size and the main difference between
PPFs is the surface compositions, the results suggest that the free
functional groups at the surface are the most crucial factor on the
enzyme immobilization efficiency.
PPF-2-NH2 presents free surface epoxide groups, due to its post-
synthetic functionalization,
which enables covalent and
irreversible conjugation with the enzyme throughout opening of
the epoxide group. On the other hand, PPF-2-CHO presents
excess of aldehyde groups on the surface, which enables
covalent conjugation via imine formation, a slow and reversible
reaction in an aqueous environment. Thus, it is expected that the
immobilization efficiency will be considerably higher for PPF-2-
NH2 than for PPF-2-CHO, due to the difference between the type
of immobilization. In fact, this significant difference of enzyme
uptake was observed and the amount of enzyme-conjugated with
PPF-2-NH2 was approximately 3 times higher than in PPF-2-CHO.
a)
b)
c)
d)
PPF-2
PPF-2
C1s
O1s
C-C/C-H
C=O
C-N/C-OH
C=N
C-O
CHO
C=O
O-H
CAL-B@PPF-2
C=O
CAL-B@PPF-2
C-C/C-H
C-N/C-OH
C-O
C=N
PPF-2 is expected to present the lowest immobilization efficiency
among the three structures. Surprisingly, its immobilization
efficiency was almost as high as PPF-2-NH2. This unexpectedly
high value could be the result of substantial non-specific
adsorption of denatured enzymes inside the pores. This view is
corroborated by the clear decrease in a specific area and pore
volume presented by PPF-2 after enzyme immobilization.
Although the immobilization efficiency is high in this case, it is
expected that the enzymatic activity will be the lowest, since most
of the enzymes present are in an inactive state.
CHO
C=O
294 292 290 288 286 284 282
Binding Energy (eV)
542 540 538 536 534 532 530 528
Binding Energy (eV)
e)
PPF-2-CHO
C1s
PPF-2-CHO
O1s
C-C/C-H
C=O
C-O
C-N/C-OH
C=N
CHO
C=O--H
O-H
CAL-B@PPF-2-CHO
C=O
CAL-B@PPF-2-CHO
C-C/C-H
C-N/C-OH
C=N
In order to evaluate the activity of the biocatalysts, a comparison
was performed among the COFs-immobilized enzymes and
Novozym 435, a commercial and widely available immobilized
enzyme, on the catalytic activity in the oleic acid esterification.
The initial rates and the specific activities are displayed in Table
3. The amount of protein in Novozym 435 was measured by the
Bradford method [48] as 30 mg of protein for each gram of support.
C-O
C=O
CHO
542 540 538 536 534 532 530 528
Binding Energy (eV)
294 292 290 288 286 284 282
Binding Energy (eV)
PPF-2-NH2
C1s
PPF-2-NH2
O1s
f)
C-C/C-H
C=O
C-N
C-O
C=N
C=O
CHO
Table 1. The relative atomic concentration of surface composition obtained
O-H
by XPS analysis of COFs and COFs-immobilized enzymes.
CAL-B@PPF-2-NH2
CAL-B@PPF-2-NH2
C-C/C-H
C=O
C-OH
C-N
C-O
C1s
79.0
70.4
77.4
69.2
83.5
73.9
N1s
7.8
O1s
10.8
17.7
16.7
19.5
8.5
N/O
0.72
0.67
0.35
0.58
0.94
0.76
C=N
C=O
CHO
PPF-2
294 292 290 288 286 284 282
Binding Energy (eV)
542 540 538 536 534 532 530 528
Binding Energy (eV)
CAL-B@PPF-2
PPF-2-CHO
11.9
5.9
Figure 3. XPS high-energy resolution spectra obtained before (PPF) and after
(CAL-B@PPF) enzyme immobilization: a) C1s of PPF-2. b) C1s of PPF-2-CHO.
c) C1s of PPF-2-NH2. d) O1s of PPF-2. e) O1s of PPF-2-CHO. f) O1s of PPF-
2-NH2.
CAL-B@PPF-2-CHO
PPF-2-NH2
11.3
8.0
The quantity and efficiency of CAL-B immobilization on COFs was
determined from the difference in enzyme concentration in the
solution before and after immobilization, and the results are
displayed in Table 2. The immobilized amount of CAL-B was
CAL-B@PPF-2-NH2
10.9
14.3
All the COFs-immobilized enzymes displayed good initial rates,
ranging from 1.8 to 4.6 mM min-1. Even though the initial rates
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10.1002/chem.201903807
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presented a lower value than that observed for Novozym 435, the
specific activities were higher for all three PPFs, ranging between
58 to 283 U/mg, 2.6 to 12.7 times greater than the observed for
Novozym 435. These results suggest that the PPF-2 has a
positive influence on the activity of CAL-B, demonstrating that
even with a smaller amount of immobilized enzyme, COFs create
an environment that enhances the overall enzymatic activity.
when the attachment of the enzyme occurs throughout
physical adsorption on the surface, which may happen in PPF-
2. This fact also corroborates the initial hypothesis that the
substantial reduction of the apparent surface area after the
enzyme immobilization in PPF-2 is due to the inclusion of
denatured proteins within the pore.
200
a)
b)
600
500
400
300
200
100
0
150
100
50
Table 2. Textural properties, enzyme loading and immobilization efficiency
on the COFs.
PPF-2
CAL-B@PPF-2
Adsorption
Desorption
Adsorption
Desorption
PPF-2-CHO
Adsorption
CAL-B@PPF-2-CHO
Adsorption
Enzyme
loading[c]
Immob.
Efficiency[d]
[a]
[b]
Desorption
Desorption
SBET
VP
0
0.0
0.0
0.2
0.4
0.6
0.8
1.0
0.2
0.4
0.6
0.8
1.0
Relative Pressure (P/Po)
Relative Pressure (P/Po)
PPF-2
1603
747
562
454
522
592
0.598
0.439
0.188
0.249
0.179
0.275
---
3.1
---
---
70
---
27
---
88
200
150
100
50
c)
CAL-B@PPF-2
PPF-2-CHO
1.2
---
CAL-B@PPF-2-CHO
PPF-2-NH2
PPF-2-NH2
Adsorption
CAL-B@PPF-2-NH2
Adsorption
Desorption
Desorption
0
0.0
0.2
0.4
0.6
0.8
1.0
3.9
CAL-B@PPF-2-NH2
Relative Pressure (P/Po)
[a] BET specific area (m2 g-1), [b] Pore volume (cm3 g-1), [c] Enzyme loading
(mg per gram of support), [d] Immobilization Efficiency (%)
Figure 4. N2 adsorption-desorption isotherms at 77K for COFs before and after
enzyme immobilization: a) PPF-2/CAL-B@PPF-2. b) PPF-2-CHO/CAL-
B@PPF-2-CHO. c) PPF-2-NH2/CAL-B@PPF-2-NH2.
It is possible to state some features of COFs that can help
elucidate this behavior. The first and most important is the
influence of the surface’s chemical characteristics on enzyme
immobilization. As shown in Table 2, in the three variations of
COFs the amount of immobilized enzyme was different, and it is
not difficult to recognize that this is a consequence of the fact that
the COF-enzyme interaction is different in all three cases.
There are other structural aspects that may play a key role in the
increase of the enzymatic activity of CAL-B when immobilized in
COFs. Firstly, the porous nature of its structure, which allows the
diffusion of the reagents throughout the support and facilitates
their access to the enzymatic site for catalysis. Secondly, its
mostly non-polar chemical nature, which creates
a
microenvironment comparable to the natural environment of CAL-
B and favors an increase in its enzymatic activity. Finnaly, beyond
the point for covalent attachment, the functionalized surface of the
COF provides multiple sites for the interaction of different residues
of the enzyme with the support, what can be achieved through
hydrophobic contacts, hydrogen bonds, and pi-stacking
interactions. This multipoint interaction in the rigid surface of the
COF can stabilize an optimal conformation for the catalytic activity
even in more drastic conditions.
Table 3. Comparison between enzymatic activity on oleic acid esterification
reaction.
Initial rate[a]
Specific activity[b]
To further explore this system, the influence of the PPFs on the
thermal stability of supported CAL-B was evaluated. The
conversions obtained in the esterification reaction between oleic
acid and ethanol, at different temperatures, are displayed in
Figure 5-a and Table 4.
Novozym® 435
6.7
1.8
3.4
4.6
22.3
58.1
CAL-B@PPF-2
CAL-B@PPF-2-CHO
CAL-B@PPF-2-NH2
283.3
117.9
a)
b)
Novozym® 435
CAL-B@PPF-2
CAL-B@PPF-2-CHO
CAL-B@PPF-2-NH2
Novozym® 435
CAL-B@PPF-2
CAL-B@PPF-2-CHO
CAL-B@PPF-2-NH2
100
75
50
25
0
100
75
50
25
0
[a] Initial rate (mM min-1), [b] Specific activity (U mg-1)
Indeed, PPF-2 and PPF-2-NH2 present fairly the same
immobilized enzyme levels, however, the specific activity of the
enzyme immobilized in PPF-2-NH2 was 2-fold higher than in
PPF-2. This difference is attributed to the post-synthetic
reaction of PPF-2-NH2 with epichlorohydrin, which promotes
the covalent attachment of the enzyme to the surface. This
could allow greater accessibility to the enzyme active site and
better maintenance of its active structure, which does not occur
40
50
60
70
0
1
2
3
4
Temperature (°C)
Number of cycles
Figure 5. a) Thermal stability and b) Recyclability test of the oleic acid
esterification reaction at 40°C.
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It is noteworthy that for all tested temperatures, the CAL-B
immobilized in the three variants of PPF-2 showed higher percent
conversions when compared to the commercial equivalent
Novozym 435 and no deactivation was observed up to 70°C. It is
known that the immobilization support could increase the thermal
stability of the biocatalyst and has been reported that especially
covalent immobilization on organic supports could prevent
deactivation in a better way than in adsorbed immobilization
method.[49]
This system was selected due to its simple and representative
model. Vinyl acetate was chosen as an acyl donor and the
reaction was carried out over a period of 2 hours with
enantiomeric excess (e.e.) values determined by chiral GC
analysis. The results are displayed in Table 5. In all cases, a high
selectivity was observed with ee values greater than 99%.
Table 5. Results for the kinetic resolution of rac-1-phenylethanol.
Table 4. Textural properties, enzyme loading and immobilization efficiency
on the PPFs.
Biocatalyst
Conversion rate[a]
e.e.[b]
>99
E[c]
Conversion rate (%)
Novozym® 435
CAL-B@PPF-2
CAL-B@PPF-2-CHO
CAL-B@PPF-2-NH2
49
16
26
32
>200
>200
>200
>200
Biocatalyst
40°C
72
50°C
65
60°C
64
70°C
73
>99
>99
>99
Novozym® 435
CAL-B@PPF-2
87
87
91
97
CAL-B@PPF-2-CHO
CAL-B@PPF-2-NH2
81
80
86
76
[a] Conversion rate (%), [b] E
nantiomeric excess (%), [c] Enantiomeric ratio
71
77
77
80
Regarding the conversions, smaller values were observed when
compared to Novozym 435. This could be attributed to the fact
that the amount of enzyme on Novozym 435 is at least 10 times
higher than in the COFs biocatalysts. Regardless of that fact,
even with a small amount of enzyme, PPF-2-CHO and PPF-2-
NH2 presented an acceptable conversion rate and an excellent
enantiomeric excess, indicating that PPFs biocatalysts hold a
potential to be applicable in the kinetic resolution process.
The thermal stability in organic solvents of free CAL-B and
Novozym 435 has been studied by Poojari and Clarson.[50] The
authors described a decrease in the relative activity of free CAL-
B from 100% at 40°C to 40% at 70°C and the enzyme showed no
catalytic activity at 90oC. Regarding to Novozym 435, only a small
decline in the relative activity was observed from 40°C to 70°C, in
agreement with our results, and relative activity of 80% was
observed at 100oC in diphenyl ether. In the case of the COFs,
even though CAL-B is exposed to the medium on the surface, the
chemical nature and structure of the support might display a key
role in the enhancement of the overall thermal stability.
Conclusions
In the present work, the application of a covalent organic
framework, reported in the literature as PPF-2, was explored as
support for enzyme immobilization. Throughout the modification
of the chemical characteristics of the surface by both synthetic
and post-synthetic methods, different approaches of enzyme
immobilization could be applied. The specific enzymatic activity
and thermal stability of the resulting biocatalyst are reported and
compared with a commercial version of immobilized enzyme –
Novozym 435. It is demonstrated that, because of their
characteristics, COFs are a suitable option as a platform for the
immobilization of enzymes, promoting an increase of the
enzymatic activity, enhancing the thermal stability, and enabling
the use of the biocatalyst throughout several cycles. The
application of the resulting biocatalyst in the kinetic resolution of
racemic mixtures is also explored, showing that this system has
an exciting potential to be explored.
Recyclability is an important feature for immobilized enzymes,
especially for industrial applications. In this way, the recyclability
of the PPFs-immobilized was studied (Figura 5-b). Regarding to
CAL-B@PPF-2, the yield of oleic acid esterification reaction for
the first cycle was 87%. In the second cycle, although a decrease
to 73% was observed, the value remained almost unaltered until
the fourth cycle. A decrease to 47% was observed in the fifth cycle.
For CAL-B@PPF-2-CHO, the yield was 82% in the first cycle,
around 70% in the second and third cycles and around 50% in the
fourth and fifth cycles. CAL-B@PPF-2-NH2 displayed a yield of
71% in the first cycle. In the second cycle, a decrease to 62% was
observed and this value remained mostly constant until the fifth
cycle. In general, the stability over several cycles has remained
remarkably close to that presented by Novozym 435, the
commercial model, indicating that the COFs biocatalysts can be
efficiently used in several cycles.
Experimental Section
Lipases are extensively used in kinetic resolution-catalyzed
enantioselective transesterification of alcohols. Particularly, CAL-
B is one of the most used lipases for the kinetic resolution, being
an environmentally friendly alternative to produce optically active
compounds. In such a manner, the kinetic resolution of rac-1-
phenylethanol by CAL-B supported in the COFs was investigated.
Tetrakis(4-aminophenyl)-methane and triformylphloroglucinol
were synthesized and purified according to the literature
procedure.[14,51] Other materials are purchased from commercial
sources.
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Synthesis of PPF-2:
A
cylindrical pressure vessel
a) Esterification conversions: GC-MS: (Shimadzu
CG2010 - DB 5 capillary column). Samples were prepared by
dissolving 10μl of the final product in 980 μL of heptane and 10
μL of MSTFA (N-Methyl-N-(trimethylsilyl) trifluoroacetamide). The
injector and detector temperatures were 250°C, and the oven
temperature was constant at 60°C for 1 min, and then increased
by 10 °C/min to 250°C, where it was held constant for 3 min.
b) Kinetic Resolution -1-phenylethanol GC-FID:
(Shimadzu CG2010 - chiral capillary column Betadex – 325) 1µL
samples were injected at 100ºC. The oven was heated at
15ºC/min to 150 ºC, at 8ºC/min to 200ºC, at 2ºC/min to 240ºC,
and then maintained for 4min. After this, the oven was heated at
15ºC/min to 300ºC.
Infrared analysis. Analysis by infrared spectroscopy used a
Shimadzu 8300 FTIR spectrophotometer. The spectrum was
obtained with 32 scans and with 4 cm−1 of resolution. For the
analysis, 10 mg of sample was placed in sample collector to form
tablets of 2 mm of thickness and 5 mm in diameter, with KBr
addition.
N2 sorption measurements: Nitrogen adsorption-desorption
isotherms were measured at 77K (temperature of liquid N2) with
Quantachrome Nova 1200 adsorption analyzer equipment.
Samples were previously degassed at 120 °C for 18 h. The
surface areas of supports and biocatalyst were determined by the
Brunauer-Emmett-Teller (BET) method.[52] Pore size distribution
curves were established from the desorption branches of the
isotherms using the non-local density functional theory (NLDFT)
model.
(Chemglass model #CG-1880-04) was charged with tetrakis(4-
aminophenyl)-methane (302.1 mg, 0.788 mmol) and
triformylphloroglucinol (222.0 mg, 1.05 mmol). The vessel was
evacuated and filled with Ar. Then it was add 15.0 ml of 1,4-
dioxane and 3.0 ml of AcOH 3M. The vessel was evacuated, filled
with Ar2 and sealed. The reaction mixture was heated at 127 °C
for 3 days to afford a yellow precipitate which was isolated by
filtration and washed with 1,4-dioxane (50 ml) and tetrahydrofuran
(50 mL). The product was soaked in tetrahydrofuran (50 mL) for
3 days. The mixture was then filtrated and after 6h under vacuum
and afforded a yellow powder (457.6 mg, 87.3%).
Synthesis of PPF-2-CHO: In a fashion similar to the
preparation of PPF-2, treatment of tetrakis(4-aminophenyl)-
methane (214.0 mg, 0.562 mmol) and triformylphloroglucinol
(316.7 mg, 1.5 mmol) in 15 mL of 1,4-dioxane and 3.0 ml of AcOH
3M at 127 °C for 3 days afforded a brown-yellow powder (467.2
mg, 88,2%) after the washing method described above.
Synthesis of PPF-2-NH2: In a fashion similar to the
preparation of PPF-2, treatment of tetrakis(4-aminophenyl)-
methane (376,5 mg, 1.0 mmol) and triformylphloroglucinol (140.5
mg, 0.667 mmol) in 15 mL of a 1,4-dioxane and 3.0 ml of AcOH
3M at 127 °C for 3 days afforded an orange powder (366.0 mg,
65.1%) after the washing method described above.
Activation: Samples of PPF-2, PPF-2-CHO and PPF-2-NH2
were loaded into a cylindrical quartz cell and heated inside a
heating mantle at a rate of 1°C/min to 120 °C under vacuum for
18 h.
Surface functionalization of PPF-2-NH2: To PPF-2-NH2
(362.0 mg) was added 11.0 mL of 1,4-dioxane and
epichlorohydrin (360.0 µL). The mixture was heated to 40°C
under stirring for 2h. The material was washed with 1,4-dioxane
and dried under vacuum, affording a bright yellow powder.
Enzyme immobilization: Supports (PPF-2, PPF-2-CHO, and
PPF-2-NH2) were subjected to the following lipase immobilization
process: 300 µL of the enzyme (CAL-B) solution (2.2 mg of protein
per mL) was dissolved in 2500 µL of 25 mM phosphate buffer pH
7.0 and added to support (150 mg). The mixture was stirred for 4
h at 40 °C using a flask shaker, before being filtered and dried
under vacuum and drying overnight at ambient temperature.
Immobilization efficiency was evaluated by the difference
between the initial amount of enzyme added and that in the
supernatant after filtration of the immobilized enzyme.
Protein loading determination. The total protein is originally
taken for immobilization and protein present in the supernatants
after immobilization was estimated by the method of Bradford[48]
using bovine serum albumin (BSA) as a standard.
Esterification reactions. Immobilized lipases (10 mg of
support in 1 mL of reaction media) were evaluated in an
esterification reaction between oleic acid and ethanol (1:1 – 100
mM in n-heptane) at different temperatures. The reactions were
performed in cryotubes under 200 rpm of agitation on a shaker.
Samples (10 μL) were collected after 1h. In order to calculate the
reaction initial velocities, reaction times were varied from 5-120
min. For thermal stability, reactions were investigated at 50–70°C.
All quantifications were determined by GC-MS analysis.
Acknowledgements
We acknowledge financial support from CAPES (Project 001),
CNPq and FAPERJ.
Keywords: Covalent Organic Framework • Enzyme •
Biocatalysis • Enantiomeric Resolution • Nanoporous Material
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Entry for the Table of Contents
FULL PAPER
Felipe L. Oliveira,[a] Stefania P. de Souza,[b]
Jonathan Bassut,[b] Heiddy M. Álvarez,[c]
Yunier Garcia-Basabe,[a] Rodrigo O. M.
Alves de Souza,[b] Pierre M. Esteves,*[a]
Raoni S. B. Gonçalves *[a]
Enzyme immobilization into COF
surface.
Page 1. – Page 8.
Enzyme-Decorated Covalent Organic
Framework as a Nanoporous Platform for
Heterogeneous Biocatalysis
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[a]
Title(s), Initial(s), Surname(s) of Author(s) including Correspond