catalysts
Article
Enantioselective Transamination in Continuous Flow
Mode with Transaminase Immobilized in a
Macrocellular Silica Monolith
Ludivine van den Biggelaar 1 , Patrice Soumillion 2 and Damien P. Debecker 1, *
1
2
*
Institute of Condensed Matter and Nanosciences, Université catholique de Louvain, Place Louis Pasteur, 1,
Box L4.01.09, 1348 Louvain-la-Neuve, Belgium; [email protected]
Institute of Life Sciences, Université catholique de Louvain, Place Croix du Sud 4-5, 1348 Louvain-la-Neuve,
Belgium; [email protected]
Correspondence: [email protected]; Tel.: +32-10-47-36-48
Academic Editor: Keith Hohn
Received: 10 January 2017; Accepted: 7 February 2017; Published: 10 February 2017
Abstract: ω-Transaminases have been immobilized on macrocellular silica monoliths and used as
heterogeneous biocatalysts in a continuous flow mode enantioselective transamination reaction.
The support was prepared by a sol-gel method based on emulsion templating. The enzyme was
immobilized on the structured silica monoliths both by adsorption, and by covalent grafting using
amino-functionalized silica monoliths and glutaraldehyde as a coupling agent. A simple reactor
set-up based on the use of a heat-shrinkable Teflon tube is presented and successfully used for the
continuous flow kinetic resolution of a chiral amine, 4-bromo-α-methylbenzylamine. The porous
structure of the supports ensures effective mass transfer and the reactor works in the plug flow regime
without preferential flow paths. When immobilized in the monolith and used in the flow reactor,
transaminases retain their activity and their enantioselectivity. The solid biocatalyst is also shown to
be stable both on stream and during storage. These essential features pave the way to the successful
development of an environmentally friendly process for chiral amines production.
Keywords: chiral amines; biocatalysis; silica monolith; enzyme immobilization; flow chemistry
1. Introduction
While the use of enzymes as catalysts in the chemical sector is extremely appealing—especially
in the perspective of sustainability—their application in industrially relevant processes is not
straightforward. Indeed, the use of free enzymes in homogeneous batch processes suffers from
severe limitations related to the impossibility to reuse and recover the biocatalysts [1]. Therefore, one
particular challenge is to make them recyclable while maintaining their activity [2]. This is typically
achieved by immobilizing the enzymes on an appropriate support—inorganic, biological, polymer or
hybrid materials [3,4].
Of particular interest nowadays, is the use of ω-transaminases for the greener production of chiral
amines, which are chiral precursors of many important drugs [5–7]. Transaminases are enzymes that
catalyze the enantioselective transfer of an amine group from an amine donor to an amino acceptor.
Genetically, engineered transaminases have demonstrated excellent performance in the synthesis
of important drug scaffolds [5]. In addition, keeping the recyclability issue in mind, researchers
have proposed successful immobilization strategies for transaminases using for example natural and
synthetic polymers [8–12] or silica [13–15] as carriers.
While enzymes immobilized on such powdery materials can be recovered by filtration or
centrifugation after use in batch reactors, a second important challenge is to perform the enzymatic
Catalysts 2017, 7, 54; doi:10.3390/catal7020054
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Catalysts 2017, 7, 54
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reaction
in a continuous
Indeed,
flow mode
allows
lowering
E-factor
of a production
process,flow
by process.
maintaining
the the
catalyst
activegenerally
over large
duration,
by the
minimizing
of
a production
process,and
by by
maintaining
thewaste/products
catalyst active ratio
over[16].
large
by minimizing
unproductive
sequences
reducing the
Toduration,
design a truly
green and
unproductive
sequences
and by
theofwaste/products
ratio
[16]. To design
a trulyisgreen
industrially practicable
process,
thereducing
conversion
a batch process to
a continuous
flow process
often
and
industrially
conversion transamination,
of a batch process
to a continuous
flow processthe
is
an essential
step practicable
[17]. In theprocess,
context the
of biocatalytic
Andrade
et al. demonstrated
often
an
essential
step
[17].
In
the
context
of
biocatalytic
transamination,
Andrade
et
al.
demonstrated
flow production of chiral amines using immobilized mutants of E. coli—designed to overexpress the
the
flow transaminase
production of [18].
chiralThis
amines
using
immobilized
mutants
of E. keeping
coli—designed
to overexpress
desired
elegant
strategy,
however,
requires
the living
organisms
the
desired
transaminase
[18].
Thisneeded
elegantnutriments
strategy, however,
alive
by feeding
them with
all the
along therequires
process.keeping the living organisms
alive Here,
by feeding
them with
all the needed nutriments
along the
we propose
the immobilization
of a transaminase
onprocess.
silica monoliths that are particularly
Here, wefor
propose
the immobilization
a transaminase
on silica
monoliths
that (where
are particularly
appropriate
the design
of continuous of
flow
reactors [19,20].
Si(HIPE)
materials
“HIPE”
appropriate
for the
design Phase
of continuous
flowwere
reactors
[19,20].
Si(HIPE)by
materials
(wheresol-gel
“HIPE”method
stands
stands for “High
Internal
Emulsion”)
recently
developed
an innovative
for
“High
Internal Phase
Emulsion”)
developed
by an innovative
sol-gel
method based
on
based
on emulsion
templating
[21]. were
Theyrecently
exhibit high
void fraction
that allows
minimizing
pressure
emulsion
templating
They exhibit
high void fraction
thatbecause
allows minimizing
pressure tableting
drop. They
drop. They
are easier[21].
to manipulate
as compared
to powders
no further shaping,
or
are
easier
to
manipulate
as
compared
to
powders
because
no
further
shaping,
tableting
or
extrusion
extrusion step is needed [22]. In addition, among the various supporting materials that can be used for
step
is needed
[22]. In addition,
the various
supporting
that can inert,
be used
for enzyme
enzyme
immobilization,
silica isamong
particularly
attractive
becausematerials
it is chemically
non-toxic
and
immobilization,
silica is particularly
attractive
because
it is chemically
inert,
andanchoring
inexpensive.
inexpensive. Moreover,
its surface can
be easily
functionalized
to allow
thenon-toxic
subsequent
of
Moreover,
surface
be easily functionalized
to allow
the subsequent
anchoring
of enzymes
enzymes its
[23].
Forcanexample,
grafting amine
groups
on silica
surface
using [23].
(3For
example, grafting amine (APTES)
groups on
silicaelectrostatic
surface using
(3-aminopropyl)triethoxysilane
aminopropyl)triethoxysilane
favors
attraction
between the silica carrier(APTES)
and the
favors
electrostatic
attraction between
the agents
silica carrier
andglutaraldehyde
the enzymes [24,25].
enzymes
[24,25]. Furthermore,
coupling
such as
can beFurthermore,
used for the coupling
covalent
agents
such as
cantheir
be used
foramino
the covalent
ofin
enzymes
through their
cross-linking
ofglutaraldehyde
enzymes through
lysine
groupscross-linking
[26–29]. Thus,
this contribution,
we
lysine
amino
groups
[26–29].Si(HIPE)
Thus, insupports
this contribution,
we make several
use of these
structuredstrategies
Si(HIPE)
make use
of these
structured
and we implement
immobilization
supports
and wethe
implement
severalofimmobilization
to demonstrate
theprocess
first example
a
to demonstrate
first example
a flow mode strategies
biocatalytic
transamination
basedofon
flow
mode biocatalytic
transamination
immobilized
transaminases
(Figure 1). process based on immobilized transaminases (Figure 1).
Figure 1.1.Transaminases
Transaminases
have
immobilized
on a macroporous
silica monolith
used as
have
beenbeen
immobilized
on a macroporous
silica monolith
and used and
as biocatalyst
biocatalyst
for continuous
flow transamination
for continuous
flow transamination
reaction. reaction.
2. Results and Discussion
2.1. Enzyme Immobilization on Si(HIPE) Monoliths
Synthesis
Si(HIPE)
support
is schematically
described
in the Supplementary
Materials
Synthesis ofofSi(HIPE)
support
is schematically
described
in the Supplementary
Materials (Figure
S1).
(Figure
Briefly, an
emulsion with
is produced
with a highofproportion
of the
dodecane
as the
dispersed
Briefly, S1).
an emulsion
is produced
a high proportion
dodecane as
dispersed
phase
and an
phase
an acidic
aqueous
continuous
phase
containing
tetraethyl orthosilicate.
Hydrolysis
and
acidic and
aqueous
continuous
phase
containing
tetraethyl
orthosilicate.
Hydrolysis and
condensation
condensation
occur
around
oillead
droplets
lead to of
thea formation
of a silica
macrocellular
reactions occurreactions
around oil
droplets
and
to theand
formation
macrocellular
scaffold. silica
After
scaffold.
After
of athe
oil phase, a monolith
self-standing
monolithfeaturing
is obtained,
featuring
specific
the removal
ofthe
theremoval
oil phase,
self-standing
is obtained,
high
specific high
surface
area
−1 ) and large void fraction (~95%). Further characterization is presented in
−1) and
surface
area
(820large
m2 ·gvoid
(820 m2·g
fraction (~95%). Further characterization is presented in Figure S2.
Figure
S2.commercial ω-transaminase (ATA-117 from Codexis) was immobilized on Si(HIPE) by three
The
methods (Figure 2). In method (a), the enzyme solution (0.2 g/L) was simply allowed to flow through
the bare silica monolith and the enzyme was immobilized by simple physical adsorption. The
Catalysts 2017, 7, 54
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The commercial ω-transaminase (ATA-117 from Codexis) was immobilized on Si(HIPE) by
three
methods
Catalysts
2017, 7, 54(Figure 2). In method (a), the enzyme solution (0.2 g/L) was simply allowed to 3flow
of 13
through the bare silica monolith and the enzyme was immobilized by simple physical adsorption.
The
monolith
is then
washed
the buffer
solution
(catalyst
denoted
“TA-Si(HIPE)”,
TA
monolith
is then
washed
with with
the buffer
solution
(catalyst
denoted
“TA-Si(HIPE)”,
where where
TA stands
stands
for
transaminase).
for transaminase).
Figure 2.
2. Immobilization
Immobilization methods:
methods: (a)
simple adsorption;
adsorption; (b)
(b) adsorption
adsorption assisted
assisted by
by electrostatic
electrostatic
Figure
(a) simple
interactions; and
and (c)
(c) covalent
covalent grafting.
grafting.
interactions;
The external surface of ATA-117 is negatively charged at the optimal working pH (pH = 8; see
The external surface of ATA-117 is negatively charged at the optimal working pH (pH = 8; see
Supplementary Materials, Figure S3). Thus, in method (b), the silica monolith was functionalized by
Supplementary Materials, Figure S3). Thus, in method (b), the silica monolith was functionalized by
APTES to bring amino functions that are positively charged at pH = 8; and the enzyme adsorption
APTES to bring amino functions that are positively charged at pH = 8; and the enzyme adsorption was
was assisted by electrostatic interactions. The initial amount of APTES introduced in the preparation
assisted by electrostatic interactions. The initial amount of APTES introduced in the preparation of
of the support was varied and the catalysts are denoted “TA-Ax”, where A stand for APTES and x is
the support was varied and the catalysts are denoted “TA-Ax”, where A stand for APTES and x is the
the APTES concentration used in the functionalization step.
APTES concentration used in the functionalization step.
ATA-117 external surface also exhibits six lysine residues (Figure S3). In method (c), glutaraldehyde
ATA-117 external surface also exhibits six lysine residues (Figure S3). In method (c), glutaraldehyde
was used as a coupling agent [26,29] to promote the covalent grafting between the amino functions
was used as a coupling agent [26,29] to promote the covalent grafting between the amino functions
on the APTES-functionalized Si(HIPE) and the lysine residues of the enzyme (catalysts denoted
on the APTES-functionalized Si(HIPE) and the lysine residues of the enzyme (catalysts denoted
“TA-Ax-GA” where GA stand for glutaraldehyde).
“TA-Ax-GA” where GA stand for glutaraldehyde).
The efficiency of APTES functionalization was verified by attenuated total reflectance infrared
The efficiency of APTES functionalization was verified by attenuated total reflectance infrared
spectroscopy (ATR-FTIR). Signals related to the amino functions are difficult to identify: N–H vibration
spectroscopy (ATR-FTIR). Signals related to the amino functions are difficult to identify: N–H vibration
at 1610 cm−1 is overlapped by water band (1600–1640 cm−1) [30]; and C–N vibration at 2259 cm−1 is on a
at 1610 cm−1 is overlapped by water band (1600–1640 cm−1 ) [30]; and C–N vibration at 2259 cm−1 is
poor diamond crystal transmittance area [31]. However, an increase in intensity of absorbance band
on a poor diamond crystal transmittance area [31]. However, an increase in intensity of absorbance
assigned to C–H stretching (from 2885 to 2974 cm−1) [30,32] is clearly observed when comparing the
band assigned to C–H stretching (from 2885 to 2974 cm−1 ) [30,32] is clearly observed when comparing
APTES-functionalized support with the pristine one (Figure 3). Expectedly, the intensity of this band
the APTES-functionalized support with the pristine one (Figure 3). Expectedly, the intensity of
increases with the APTES concentrations used for functionalization. Compared to the IR spectrum of pure
this band increases with the APTES concentrations used for functionalization. Compared to the IR
APTES, the spectra of APTES-functionalized Si(HIPE) samples show a more intense band for CH2
spectrum of pure APTES, the−1spectra of APTES-functionalized Si(HIPE) samples show a more intense
vibrations (2855 and 2926 cm ) and less intense band for CH3 stretching vibrations (2885 and 2974 cm−1)
band for CH2 vibrations (2855 and 2926 cm−1 ) and less intense band for CH3 stretching vibrations
[30,32]. This indicates that ethoxy groups of APTES are eliminated during functionalization, consistent
(2885 and 2974 cm−1 ) [30,32]. This indicates that ethoxy groups of APTES are eliminated during
with the silanization process where ethoxy groups react with the silanol groups of the support.
functionalization, consistent with the silanization process where ethoxy groups react with the silanol
Thermogravimetric analyses confirmed the presence of additional organic species in the APTES
groups of the support.
functionalized samples (Figure 4). A relatively well-defined weight loss is observed between 300 and
Thermogravimetric analyses confirmed the presence of additional organic species in the APTES
600 °C with the functionalized samples (and not with the pristine Si(HIPE)). It is thus assigned to
functionalized samples (Figure 4). A relatively well-defined weight loss is observed between 300 and
aminopropyl moiety decomposition [33,34]. The weight loss is larger when the APTES concentration
600 ◦ C with the functionalized samples (and not with the pristine Si(HIPE)). It is thus assigned to
used to functionalize the silica monolith is higher.
aminopropyl moiety decomposition [33,34]. The weight loss is larger when the APTES concentration
In principle, the enzyme loading in each monolith can be evaluated by a simple mass
used to functionalize the silica monolith is higher.
balance between the solution used to load the monolith and the recovered solution, including the
In principle, the enzyme loading in each monolith can be evaluated by a simple mass balance
washing solution. Unfortunately, these measurements suffered from high variability and discrepancies,
between the solution used to load the monolith and the recovered solution, including the washing
which prevent us from providing the precise enzyme loading for each monolith (as discussed in the
solution. Unfortunately, these measurements suffered from high variability and discrepancies,
Supplementary Materials, Figures S4 and S5). As a general trend, however, TA-Si(HIPE) and TA-Ax
which prevent us from providing the precise enzyme loading for each monolith (as discussed in
with low APTES content showed low enzyme loading values, certainly below 0.5 mg of enzyme in the
the Supplementary Materials, Figures S4 and S5). As a general trend, however, TA-Si(HIPE) and TA-Ax
monolith. For the TA-Ax-GA catalysts with high APTES content, the loading is clearly higher and it
is possible to provide an estimation of the enzyme loading. In TA-A50-GA—the most active catalyst,
vide infra—enzyme loading was estimated at 1.2 mg.
Catalysts 2017, 7, 54
4 of 13
with low APTES content showed low enzyme loading values, certainly below 0.5 mg of enzyme in the
monolith. For the TA-Ax-GA catalysts with high APTES content, the loading is clearly higher and it is
possible to provide an estimation of the enzyme loading. In TA-A50-GA—the most active catalyst, vide
infra—enzyme
loading was estimated at 1.2 mg.
Catalysts 2017, 7, 54
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Figure 3. ATR-FTIR spectra of: (a) pure APTES ((3-aminopropyl)triethoxysilane); (b) TA-A50; (c) TA-
Figure 3. ATR-FTIR spectra of: (a) pure APTES ((3-aminopropyl)triethoxysilane);
(b) TA-A50;
A10; and (d) Si(HIPE). An increase in C–H stretching band absorbance (around 2900 cm−1) suggests a
(c) TA-A10;
and
(d)
Si(HIPE).
An
increase
in
C–H
stretching
band
absorbance
(around
2900 cm−1 )
higher amount of aminopropyl groups grafted to monolith surface for higher APTES concentrations.
suggests a higher amount of aminopropyl groups grafted to monolith surface for higher
Figure 3. ATR-FTIR spectra of: (a) pure APTES ((3-aminopropyl)triethoxysilane); (b) TA-A50; (c) TAAPTES concentrations.
−1
A10; and (d) Si(HIPE). An increase in C–H stretching band absorbance (around 2900 cm ) suggests a
higher amount of aminopropyl groups grafted to monolith surface for higher APTES concentrations.
Figure 4. Relative organic content of Si(HIPE), TA-A10 and TA-A50 samples measured by TGA
(relative mass loss over 150 °C).
2.2. Transaminations in Continuous Flow Mode
The 4.kinetic
of ofracemic
(rac-BMBA)
Figure
Relative resolution
organic content
Si(HIPE), bromo-α-methylbenzylamine
TA-A10 and TA-A50 samples measured
by TGAinto
Figure 4. Relative organic content of Si(HIPE), TA-A10 and TA-A50 samples measured by TGA (relative
4’-bromoacetophenone
was studied as a model transamination reaction to evaluate the
(relative mass
loss over(BAP)
150 °C).
mass loss
over 150of◦the
C). solid biocatalysts (Figure 5). The thermodynamic equilibrium for this reaction is
performance
highly
favorable and
conversionFlow
of ~99%
2.2.
Transaminations
in aContinuous
Modeis expected at equilibrium [35,36]. However, without any
catalyst,
this
conversion
is
very
slow
(see
Supplementary Materials, Figure S6). In the presence of
2.2. Transaminations
in Continuous
The kinetic
resolutionFlow
of Mode
racemic bromo-α-methylbenzylamine (rac-BMBA) into
ATA-117, a R-selective transaminase, R-BMBA conversion into BAP is strongly catalyzed. In this paper,
4’-bromoacetophenone (BAP) was studied as a model transamination reaction to evaluate the
yield is defined
as the proportion
of rac-BMBA
converted to 4’-bromoacetophenone (BAP;
in %).
The the
kinetic
resolution
of racemic
bromo-α-methylbenzylamine
(rac-BMBA)
into
performance
of the
solid
biocatalysts
(Figure 5).
The thermodynamic
equilibrium for this reaction is
The
maximum
yield
for
the
kinetic
resolution
is
thus
50%.
4’-bromoacetophenone
(BAP)
was
studied
as
a
model
transamination
reaction
to
evaluate
the
highly favorable and a conversion of ~99% is expected at equilibrium [35,36]. However, without any
performance
of
the
solid
biocatalysts
(Figure
5).
The
thermodynamic
equilibrium
for
this
reaction
is
catalyst, this conversion is very slow (see Supplementary Materials, Figure S6). In the presence of
ATA-117, aand
R-selective
transaminase,
R-BMBA
conversionat
into
BAP is strongly
catalyzed.
In this paper,
highly favorable
a conversion
of ~99%
is expected
equilibrium
[35,36].
However,
without any
yield
is defined as
proportion
of rac-BMBA
converted Materials,
to 4’-bromoacetophenone
in presence
%).
catalyst, the
this
conversion
is the
very
slow (see
Supplementary
Figure S6). (BAP;
In the
of
The maximum yield for the kinetic resolution is thus 50%.
ATA-117, a R-selective transaminase, R-BMBA conversion into BAP is strongly catalyzed. In this paper,
the yield is defined as the proportion of rac-BMBA converted to 4’-bromoacetophenone (BAP; in %).
The maximum yield for the kinetic resolution is thus 50%.
Catalysts 2017, 7, 54
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Figure 5. Flow
Flow kinetic
kinetic resolution
resolution of
of rac-BMBA
rac-BMBA by
by ATA-117
ATA-117 transaminases immobilized
immobilized on
on Si(HIPE)
Si(HIPE)
monolith. As
As ATA-117
ATA-117 is R-selective, it only accepts the R-BMBA as a substrate, leaving the S-BMBA
S-BMBA
monolith.
unreacted. PLP stands for Pyridoxal phosphate, the transaminase cofactor.
Figure 5. Flow kinetic resolution of rac-BMBA by ATA-117 transaminases immobilized on Si(HIPE)
monolith.
ATA-117
R-selective,
itin
only
accepts the R-BMBA
asin
a substrate,
leaving
the S-BMBA
The
isisAs
carried
in isflow
mode
ainhomemade
set-up
which
the
monolith
is casted
into a
The reaction
reaction
carried
mode
a homemade
set-up
in which
the monolith
is casted
unreacted.
PLP
stands in
for flow
Pyridoxal
phosphate,
the transaminase
cofactor.
heat-shrinkable
Teflon tube
andtube
connected
to a syringe
at onepump
side and
to a side
sample
collector
at the
into a heat-shrinkable
Teflon
and connected
topump
a syringe
at one
and
to a sample
other
side
(Figures
6
and
S7).
The
set-up
and
the
structure
of
the
support
prepared
by
the
emulsion-based
is carried
in flow6 mode
in a homemade
set-up
in which
thethe
monolith
is casted
collector at The
thereaction
other side
(Figure
and Figure
S7). The
set-up
and
structure
of into
the asupport
sol-gel
method
a plug
flow
regime
through
the
macroporous
support
(Figure
heat-shrinkable
Teflon
tube and
connected
to
a syringe
pump
at oneflow
side and
to a sample
collector
at Relatively
the
prepared
by theensured
emulsion-based
sol-gel
method
ensured
a plug
regime
through
theS8).
macroporous
other
side
(Figures
6
and
S7).
The
set-up
and
the
structure
of
the
support
prepared
by
the
emulsion-based
short
contact
times
selected
to remain
the were
initialselected
activitytoregime
conversion).
support
(Figure
S8).were
Relatively
short
contact in
times
remain(low
in the
initial activity regime
sol-gel method ensured a plug flow regime through the macroporous support (Figure S8). Relatively
(low conversion).
short contact times were selected to remain in the initial activity regime (low conversion).
Figure 6. Si(HIPE) monolith placed in a heat-shrinkable PTFE tube. Monolithic reactor is connected
Figure 6.
Si(HIPE) monolith placed in a heat-shrinkable PTFE tube. Monolithic reactor is connected to
to syringe-pump and immerged in a water bath.
Figure
6. Si(HIPE)
in a bath.
heat-shrinkable PTFE tube. Monolithic reactor is connected
syringe-pump
and monolith
immergedplaced
in a water
to syringe-pump
and
immerged
in
a
water
bath.
The pristine Si(HIPE) and the APTES
functionalized supports showed no catalytic activity
(Figure S9). The pristine Si(HIPE) monolith was initially tested as a support for the transaminase (sample
The pristine Si(HIPE) and the APTES functionalized supports showed no catalytic activity
TheTA-Si(HIPE)).
pristine Si(HIPE)
the APTES
functionalized
supports
showed
no catalytic
The yield and
was moderate,
decreased
in the first hour
of reaction,
and stabilized
aroundactivity
(Figure 1.1%
S9). (Figure
The pristine
Si(HIPE)
monolith
was initially
tested
a support
for
transaminase
7). Initial
deactivation
maywas
tentatively
be tested
attributed
aaspartial
leaching
of the
the enzyme
(Figure S9).
The pristine
Si(HIPE)
monolith
initially
as atosupport
for
the transaminase
(sample
(sampleout
TA-Si(HIPE)).
The
yield
was
moderate,
decreased
in
the
first
hour
of
reaction,
and
of the
monolith.
Functionalization
of the surface
perreaction,
method (b)—is
expectedstabilized
toaround
TA-Si(HIPE)).
The
yield was
moderate, decreased
inwith
the APTES—as
first hour of
and stabilized
around modify
1.1% (Figure
7).charge
Initialofdeactivation
may
tentatively
attributed
to a partial
the surface
the silica support
and
to favor the be
electrostatic
adsorption
of theleaching
enzyme of the
1.1% (Figure 7). Initial deactivation may tentatively be attributed to a partial leaching of the enzyme
on
the
support.
Indeed,
higher
conversion
was
obtained
with
TA-A10,
but
the
activity
level
was not (b)—is
enzyme out of the monolith. Functionalization of the surface with APTES—as per method
out of the
monolith.
Functionalization
ofa true
the surface
with
APTES—as
per method
(b)—is
expected
to
very
stable
with
time.
In
method
(c),
covalent
anchoring
of
the
enzyme
is
obtained
under
the
expected to modify the surface charge of the silica support and to favor the electrostatic adsorption
of
modify action
the surface
charge of the
silica
supportagent
andbetween
to favorthe
theamine
electrostatic
of glutaraldehyde
used
as coupling
functionsadsorption
incorporatedofonthe
theenzyme
the enzyme on the support. Indeed, higher conversion was obtained with TA-A10, but the activity level
on the support.
higher
was obtained
with TA-A10,
but the
level was not
surface ofIndeed,
the support
and conversion
the amine functions
of the enzyme.
Pre-activation
withactivity
glutaraldehyde
was not(TA-A10-GA)
very stable with
time.
In method
(c),
a true
covalent
of the enzyme is obtained under
indeed
allowed
reaching
directly
a stable
yieldanchoring
of 2.1%.
very stable with time.
In method
(c), a true
covalent
anchoring
of the enzyme is obtained under the
the action ofInglutaraldehyde
used
as coupling
agent between
the amine functions
incorporated on the
an attempt to further
increase
the reaction
the APTES
in the functionalization
action of glutaraldehyde
used as
coupling
agentyield,
between
the concentration
amine functions
incorporated on the
was raisedand
to 20the
andamine
50 mM,functions
keeping all of
other
The yield with
increased
when
surface solution
of the support
theparameters
enzyme.constant.
Pre-activation
glutaraldehyde
surface of the support and the amine functions of the enzyme. Pre-activation with glutaraldehyde
the concentration
of APTESreaching
used for functionalization
increased
(Figure
(TA-A10-GA)
indeed allowed
directly a stable
yield of
2.1%.8), expectedly suggesting that
(TA-A10-GA)
a stable
yield
of 2.1%.
higherindeed
density allowed
of grafted reaching
APTES on directly
the monolith
surface
is leading
to a higher enzymatic loading.
In aan
attempt
to
further
increase
the reaction
yield,
the
APTES concentration in the
In an
to further
increase
thefor
reaction
yield,sample
the APTES
thebeen
functionalization
Theattempt
highest BAP
yield was
obtained
TA-A50-GA
(6.2%).concentration
The experimentin
has
carried
functionalization
solutionshowed
was raised
torepeatability
20 and 50 mM,
keeping
all other parameters
constant.
The yield
three
timestoand
goodkeeping
(relative
standard deviation
9%).
solutionout
was
raised
20 and 50 amM,
all other
parameters
constant.(RSD)
The was
yield
increased when
increased when the concentration of APTES used for functionalization increased (Figure 8), expectedly
the concentration of APTES used for functionalization increased (Figure 8), expectedly suggesting that
suggesting that a higher density of grafted APTES on the monolith surface is leading to a higher
a higher density of grafted APTES on the monolith surface is leading to a higher enzymatic loading.
enzymatic loading. The highest BAP yield was obtained for TA-A50-GA sample (6.2%). The experiment
The highest BAP yield was obtained for TA-A50-GA sample (6.2%). The experiment has been carried
has been carried out three times and showed a good repeatability (relative standard deviation (RSD)
out three times and showed a good repeatability (relative standard deviation (RSD) was 9%).
was 9%).
Catalysts 2017, 7, 54
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2017, 7, 54
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6 of 13
7. APTES and glutaraldehyde pre-activation effects on biocatalysts efficiency: (■) Si(HIPE); (●)
Figure
7. APTES
and
glutaraldehyde
pre-activationeffects
effectson
onbiocatalysts
biocatalystsefficiency:
efficiency: (■)
Figure
7. Figure
APTES
and
glutaraldehyde
pre-activation
( ) Si(HIPE);
Si(HIPE); (●)
TA-A10; (▲) TA-A10-GA; and (◆), reagent solution (containing rac-BMBA).
(
N
)
TA-A10-GA;
and
(
),
reagent
solution
(containing
rac-BMBA).
TA-A10;
(▲)
TA-A10-GA;
and
(◆),
reagent
solution
(containing
rac-BMBA).
s efficiency: (■) Si(HIPE); (●)) TA-A10;
Figure 7. APTES and glutaraldehyde pre-activation effects on biocatalysts efficiency: (■) Si(HIPE); (●)
A).
TA-A10; (▲) TA-A10-GA; and (◆), reagent solution (containing rac-BMBA).
Figure 8. Effect of APTES concentration in the grafting solution on biocatalysts efficiency. Increasing
APTES concentration allows enhancing the yield. Yield values are the averaged yield measured after
180 min time on stream (where the yield appeared to stabilize).
This level of conversion was achieved with a 10 min contact time. If the flow rate and the size of
Figure
8. Effect
of APTES
concentrationin
inthe
the grafting
grafting solution
on biocatalysts
efficiency.
Increasing
Figure
8.monolith
Effect
ofare
APTES
concentration
solution
biocatalysts
efficiency.
Increasing
the
in parallel while
keeping
the
contact
timeon
constant,
the yield
remains
at
theIncreasing
Figure
Effect
ofvaried
APTES
the Yield
grafting
solution
biocatalysts
efficiency.
APTES8.concentration
allowsconcentration
enhancing the in
yield.
values
are the on
averaged
yield measured
after
APTES
concentration
enhancing
yield.
Yield values
are theisaveraged
yield
measured
after
same
level (Figureallows
S10). This
confirmsthe
that
the activity
measurement
not biased
by diffusional
enhancing
the yield.
Yield values are the averaged yield measured after
talysts efficiency. Increasing APTES
180
minconcentration
time onaddition,
streamallows
(where
theofyield
appeared
to stabilize).
limitations.
the level
conversion
is far
enough from the thermodynamic equilibrium
180 min
time on In
stream (where
the yield
appeared
to stabilize).
time on stream
(where theofyield
appeared
to stabilize).
eraged yield measured after 180
to min
be considered
as a measurement
the initial
activity.
Indeed, conversion is roughly divided by
This
level
of conversion
achieved
withisarun
10 min
time. If the
flowtow
ratemonoliths,
and the size of
two or
multiplied
by two was
when
the reaction
withcontact
half a monolith
or with
This
level
ofare
conversion
was
achieved
with
athe
10
min
contact
time.
flow
rate
the
the This
monolith
varied
in parallel
while
keeping
contact
time
constant,
yield
remains
at the
respectively
S11).
level
of(Figure
conversion
was
achieved
with
a 10
min
contact
time.Ifthe
Ifthe
the
flow
rateand
and
thesize
size of
Looking
at S10).
the rough
estimation
ofthat
thekeeping
enzyme
loading
in this monolith
(~1.2
mg), the
it isby
possible
same
level
(Figure
This
confirms
the activity
not biased
diffusional
of the
the
monolith
are
in
parallel
while
the measurement
contact
constant,
the
yield
remains at
at the
the
monolith
are varied
varied
in
parallel
while
time is
constant,
yield
remains
If the flow rate and
size
of
to estimate
the residual
activity
of conversion
the immobilized
enzyme,
asfrom
compared
to the free enzyme.
The
limitations.
In
addition,
the
level
of
is
far
enough
the
thermodynamic
equilibrium
the
same
level
(Figure
S10).
This
confirms
that
the
activity
measurement
is
not
biased
by
diffusional
same
S10). This confirms that the activity measurement is not biased by diffusional
stant, the yield remains
at the
obtained
in flow (6.2%)ofis the
compared
to
the conversion
ofconversion
a homogeneous
batch reactor
to be conversion
considered
as athe
measurement
initial
activity.
Indeed,
is roughly
divided
by
In
addition,
level
ofenzyme
conversion
is far
from
the
thermodynamic
equilibrium
to
limitations.
In
addition,
the level
of conversion
isenough
far enough
from
the equal
thermodynamic
equilibrium
t is not biased bylimitations.
diffusional
containing the same amount
of
in the same
conditions
and
after
a period
to the contact
two or multiplied by two when the reaction is run with half a monolith or with tow monoliths,
considered
measurement
of theininitial
activity.
Indeed, in
conversion
isresidual
roughly
two or by
time. as
Thea ratio
conversion
flow
over
the conversion
batch is the
activity.
The bydivided
to be considered
asofa the
measurement
of the
initial
activity.
Indeed,
conversion
isdivided
roughly
e thermodynamic be
equilibrium
respectively (Figure S11).
equivalent
homogeneous
batch
give
a 37.5%
inwith
the same
conditions,
so monoliths,
thator
thewith
residual
by two when
the
reaction
is
run
with
half
a monolith
or
with
tow
respectively
two orLooking
multiplied
two
whenshould
the
isyield
run
half
a monolith
tow
monoliths,
version is roughlymultiplied
divided
by
the by
rough
estimation
of reaction
the enzyme
loading
in this
monolith
(~1.2 mg),
it is possible
activity isat
estimated
to 16%
in this case.
S11).
respectively
(Figure
S11).
nolith or with tow(Figure
monoliths,
to estimate the residual activity of the immobilized enzyme, as compared to the free enzyme. The
Looking
atobtained
the
rough
estimation
ofcompared
the
enzyme
loading
in this
monolith
(~1.2
mg),
itreactor
isitpossible
Looking
at the
rough
estimation
of the
enzyme
loading
inofthis
monolith
(~1.2
mg),
is possible
conversion
in flow
(6.2%) is
to the
conversion
a homogeneous
batch
toisestimate
thethe
residual
activity
ofofthe
enzyme,
compared
to the
the
free
enzyme.The
to
estimate
the
residual
activity
the
immobilized
enzyme,
as compared
to
free
enzyme.
containing
same
amount
of enzyme
inimmobilized
the
same conditions
and after
a period equal
to the
contact
onolith (~1.2 mg), it
possible
time.The
The ratio
of thein
conversion
in flow
the conversion
in
batch isofthe
residual
activity.batch
Thebatch
Theconversion
conversion
obtained
in
flow
(6.2%)
isover
compared
to the
conversion
a homogeneous
obtained
flow
(6.2%)
is compared
to the
conversion
aofhomogeneous
reactor
pared to the free enzyme.
equivalent
homogeneous
batch
should
give
a
37.5%
yield
in
the
same
conditions,
so
that
the
residual
reactor
containing
the
same
amount
of
enzyme
in
the
same
conditions
and
after
a
period
equal
to
the
containing
a homogeneous batch
reactor the same amount of enzyme in the same conditions and after a period equal to the contact
activity
is
estimated
to
16%
in
this
case.
time.
of the
conversion
in flow
over
conversion
batch
residual
activity.The
time.
The The
ratioratio
of the
conversion
in flow
over
thethe
conversion
in in
batch
is is
thethe
residual
activity.
r a period equal tocontact
the
contact
Theequivalent
equivalent
homogeneous
batch
should
give
a 37.5%
yield
in same
the same
conditions,
so the
thatresidual
the
batch
should
give
a 37.5%
yield
in the
conditions,
so that
h is the residual activity.
The homogeneous
residual
activity
is
estimated
to
16%
in
this
case.
onditions, so that theactivity
residualis estimated to 16% in this case.
Catalysts 2017, 7, 54
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7 of 13
7 of 13
2.3. Applicability
Applicability and
and Robustness
Robustness of the Biocatalytic Flow Reactor
A specific experiment was carried out to demonstrate that the enzyme remains enantioselective
when immobilized (Figure 9). In the first
first part of the
the experiment,
experiment, only
only S-BMBA
S-BMBA was
was fed
fed in
in the
the reactor
reactor
(instead
of
the
racemic
BMBA).
As
expected,
the
BAP
yield
remain
very
low
at
the
reactor
flow
output,
(instead of the racemic BMBA). As expected, the BAP yield remain very low at the reactor flow
corresponding
exactly to
the non-catalytic
conversion
occurring
in the in
reagent
solution
in theinsole
output, corresponding
exactly
to the non-catalytic
conversion
occurring
the reagent
solution
the
presence
of PLP.
180 min,
racemic
BMBABMBA
solution
was injected
in the reactor.
a 140-min
sole presence
of After
PLP. After
180the
min,
the racemic
solution
was injected
in the After
reactor.
After a
lag
time required
time for the
medium medium
to pass through
the flowthe
device,
yield
reaches
140-min
lag time required
timereaction
for the reaction
to pass through
flowBAP
device,
BAP
yield
6%
again.
demonstrates
unambiguously
that ATA-117
immobilized
in the flow
reactor
reaches
6%This
again.
This demonstrates
unambiguously
that ATA-117
immobilized
in the
flowremains
reactor
enantioselective.
remains enantioselective.
Figure 9. TA-A50-GA
TA-A50-GA enantioselectivity.
enantioselectivity. With S-BMBA enantiomer, the yield is low and corresponds
corresponds
to the
the non-enzymatic
non-enzymatic reaction. When the racemic BMBA is injected in the reactor at 180 min, the
the yield
yield
raises up to 6% after 2 h, which is the time required for the solution to reach the output.
output.
From
is important
to ensure
thatthat
the biocatalyst
allows
reaching
total
From an
anindustrial
industrialperspective,
perspective,it it
is important
to ensure
the biocatalyst
allows
reaching
conversion,
i.e.,
the
actual
kinetic
resolution
with
only
one
BMBA
enantiomer
remaining
at
total conversion, i.e., the actual kinetic resolution with only one BMBA enantiomer remaining at the
the
output.
To
that
end,
eight
functionalized
monoliths
were
connected
in
series
and
then
pre-activated
output. To that end, eight functionalized monoliths were connected in series and then pre-activated
with
(TA-A50-GA
samples,
Figure
10).10).
After
a 200-min
lag
with glutaraldehyde
glutaraldehydeand
andloaded
loadedwith
withthe
theenzyme
enzyme
(TA-A50-GA
samples,
Figure
After
a 200-min
time
required
time
for
the
reaction
medium
to
pass
fully
through
the
flow
device,
the
yield
reached
lag time required time for the reaction medium to pass fully through the flow device, the yield reached
the
the maximum
maximum of
of 50%.
50%. R-BMBA
R-BMBA was
was fully
fully converted,
converted, as
as attested
attested by
by chiral
chiral HPLC.
HPLC.
To
assess
storage
stability
and
recyclability,
a
TA-A50-GA
biocatalyst
tested
a classical
To assess storage stability and recyclability, a TA-A50-GA biocatalyst waswas
tested
in a in
classical
flow
flow
reaction,
which
was
interrupted
after
4
h.
The
reactor
was
sealed
at
both
ends
and
stored
4 for
°C
◦C
reaction, which was interrupted after 4 h. The reactor was sealed at both ends and stored at 4at
for
16Then,
h. Then,
reactor
was
again
put
placeininthe
theflow
flowset-up
set-upand
andfresh
fresh reaction
reaction medium
16 h.
thethe
reactor
was
again
put
ininplace
medium was
was fed.
fed.
The
yield
was
practically
as
high
as
before
cold
storage
(~5.5%
instead
of
~6%;
Figure
11).
After
The yield was practically as high as before cold storage (~5.5% instead of ~6%; Figure 11). After 55 hh of
of
reaction,
the
reactor
was
again
removed,
sealed
and
stored
at
4
°C
for
68
h.
Then,
it
was
tested
again
◦
reaction, the reactor was again removed, sealed and stored at 4 C for 68 h. Then, it was tested again
with
medium and
and the
the final
final yield
yield was
was still
still ~4.7%.
~4.7%. Thus,
with fresh
fresh reaction
reaction medium
Thus, residual
residual activity
activity was
was 80%
80% after
after
four
days
of
cold
storage,
which
indicates
a
good
stability
and
recyclability
of
the
biocatalysts.
four days of cold storage, which indicates a good stability and recyclability of the biocatalysts.
Catalysts 2017, 7, 54
8 of 13
Catalysts 2017, 7, 54
8 of 13
Figure 10.
10. (Top)
(Top)Reactor
Reactor
made
of eight
TA-A50-GA
samples
ininseries
ordertotal
to reach
total
made
of eight
TA-A50-GA
samples
in series
order in
to reach
conversion.
Figure 10.
(Top)
Reactor
of by
eight
TA-A50-GA
samples
incontact
series
in order
toTA-A50-GA
reach
total
conversion.
(Bottom)
Total
conversion
is reached
by increasing
time
(eight
(Bottom)
Total
conversion
ismade
reached
increasing
contact
time (eight
TA-A50-GA
samples
in series).
conversion.
(Bottom) Total conversion is reached by increasing contact time (eight TA-A50-GA
samples
in series).
samples in series).
Figure
11. Yield
with TA-A50-GA,
flow reaction
is carried
out for out
periods
of ~14 hofinterrupted
by cold storage.
Figure
11. Yield
with TA-A50-GA,
flow reaction
is carried
for periods
~14 h interrupted
by
Figure
11.
Yield
with
TA-A50-GA,
flow
reaction
is
carried
out
for
periods
of
~14
h
interrupted
by cold storage.
cold storage.
3. Materials and Methods
3. Materials
Methods
3.
Materials and
and Methods
3.1. Materials
3.1. Materials
Acetone (≥99.9%), (3-aminopropyl)triethoxysilane (APTES; ≥98%), Brilliant blue G 250,
Acetone ((≥99.9%),
≥99.9%),(BAP;
(3-aminopropyl)triethoxysilane
(APTES; ≥98%),
≥98%),
Brilliant
blue
G 250,
(3-aminopropyl)triethoxysilane
(APTES;
Brilliant
blue
G
4’-bromoacetophenone
≥ 98%), R-4-bromo-α-methylbenzylamine
(R-BMBA,
99%),
S-4-bromo4’-bromoacetophenone
(BAP;
≥
98%),
R-4-bromo-α-methylbenzylamine
(R-BMBA,
99%),
S-4-bromo-α4’-bromoacetophenone
(BAP;
≥
98%),
R-4-bromo-α-methylbenzylamine
(R-BMBA,
99%),
S-4-bromoα-methylbenzylamine (S-BMBA, 99%), dodecane (≥90%), hydrochloric acid (37% wt, aqueous solution),
methylbenzylamine
(S-BMBA,
99%),
dodecane
(≥
90%), hydrochloric
(37%
wt, sodium
aqueoushydroxide
solution),
α-methylbenzylamine
(S-BMBA,
99%),
dodecane
(≥90%),
hydrochloric
acid
(37%
phosphoric
acid aqueous
solution
(85%),
pyridoxal
5’-phosphate
hydrateacid
(PLP;
≥98%),
phosphoric
acid
(85%),
pyridoxal
5’-phosphate
hydrate
(PLP; ≥98%),
hydroxide
phosphoric
acidaqueous
aqueous
solution
(85%),
pyridoxal
5’-phosphate
hydrate
(PLP;sodium
≥≥98%),
98%),
sodium
aqueous
solution
(50%),solution
sodium
pyruvate
(≥99%),
tetraethyl
orthosilicate
(TEOS;
toluene
aqueous
solution
(50%),
sodium
pyruvate
(≥99%),
tetraethyl
orthosilicate
(TEOS;
≥98%),
toluene
hydroxide
aqueous
solution
(50%),
sodium
pyruvate
(
≥
99%),
tetraethyl
orthosilicate
(TEOS;
≥98%),
(≥99.8%, anhydrous) and trimethyltetradecylammonium bromide (TTAB; ≥99%) were purchased
from
(≥99.8%, (anhydrous)
and trimethyltetradecylammonium
bromide (TTAB;
≥99%) (TTAB;
were≥99%;
purchased
from
toluene
≥99.8%,
anhydrous)
and trimethyltetradecylammonium
bromide
≥99%)
were
Sigma-Aldrich
(Darmstadt,
Germany).
4-bromo-α-methylbenzylamine
(rac-BMBA;
racemate),
Sigma-Aldrich
(Darmstadt,
Germany).
4-bromo-α-methylbenzylamine
(rac-BMBA;
≥99%;
racemate),
dibasic potassium phosphate (≥99%), dimethylsulfoxide (DMSO; ≥99.8%) and monobasic potassium
dibasic potassium phosphate (≥99%), dimethylsulfoxide (DMSO; ≥99.8%) and monobasic potassium
Catalysts 2017, 7, 54
9 of 13
purchased from Sigma-Aldrich (Darmstadt, Germany). 4-bromo-α-methylbenzylamine (rac-BMBA;
≥99%; racemate), dibasic potassium phosphate (≥99%), dimethylsulfoxide (DMSO; ≥99.8%)
and monobasic potassium phosphate (≥99%) were purchased from Acros (Geel, Belgium).
Dichloromethane (≥99.80%), diethylamine (≥99%), isohexane (HPLC grade), 2-propanol (HPLC grade)
and tetrahydrofuran (THF; ≥99.6%) were purchased from VWR (Leuven, Belgium). Heat-shrinkable
polytetrafluoroethylene (PTFE) tube was purchased from RS Components (Brussels, Belgium).
Transaminase ATA-117 was generously supplied by Codexis (Redwood, CA, USA). Distilled water
was applied for all synthesis and treatment processes.
3.2. Support Synthesis—Si(HIPE) Monolith
Porous monoliths synthesis was performed according to Ungureanu et al. [21] (see Supplementary
Materials, Figure S1): 6 g of a concentrated hydrochloric acid solution (37% wt) were introduced in
16 g of a TTAB aqueous solution (35% wt). Then 5 g of TEOS was added. The aqueous phase was
stirred until a monophasic hydrophilic medium was obtained. To form an emulsion, 35 g dodecane
was then added dropwise in a mortar while stirring. Emulsion was cast into a polypropylene 10 mL
flask, allowed to condense for 1 week at room temperature. Resulting material was washed three times
with 50 mL of a THF/acetone mixture (1:1 v/v) (each washing lasted 24 h) and then gently dried in air
during 3 days before calcined at 650 ◦ C for 6 h (heating rate of 2 ◦ C/min with a first plateau at 180 ◦ C
for 6 h). Porous monoliths were stored in a desiccator at room temperature.
3.3. Functionalization of the Monolith Surface
Samples are denoted Ax where A stands for APTES grafting and x is the concentration in
APTES (mM) in the grafting solution. This protocol was adapted from Ungureanu et al. [21]:
2 monoliths (approximately 0.15 g each) were added into 25 mL of a toluene/APTES solution of
desired concentration. Dynamic vacuum was applied to force the solution into the pores of the
monoliths, until effervescence stopped. Static vacuum was then maintained for 24 h. Monoliths were
separated from the toluene/APTES solution and washed three times with 25 mL of a toluene/acetone
mixture (1:1 v/v) by applying dynamic vacuum until effervescence stopped and static vacuum for
2 h. Hybrid monoliths were dried under vacuum at 60 ◦ C for 24 h and then stored in a desiccator at
room temperature.
3.4. Characterizations
3.4.1. Attenuated Total Reflectance—InfraRed spectroscopy (ATR-FTIR)
Samples were analyzed using a Bruker Equinox 55 (Bruker, Brussels, Belgium) with a Platinum
ATR cell, with a diamond crystal, and Trans DTGS detector. One hundred scans were taken for both
background and samples, with a resolution of 2 cm−1 . ATR correction was applied (number of ATR
reflection is 1; angle of incidence is 45◦ ; mean reflection index of sample is 1.5). Monoliths were
systematically crushed.
3.4.2. Thermo Gravimetric Analysis (TGA)
Samples were dried under vacuum at 105 ◦ C for 24 h. TGA was performed under air flow
(100 mL/min) with heating rate of 10 ◦ C/min from 25 ◦ C until 900 ◦ C with a TGA/SDTA-851e Mettler
Toledo equipment (Mettler Toledo, Zaventem, Belgium).
3.4.3. Scanning Electron Microscopy (SEM)
SEM micrographs were taken with a JEOL 7600F (JEOL, Zaventem, Belgium) operated at a 15.0 kV
voltage. Samples were dried under vacuum at 60 ◦ C for 24 h and then placed on a piece of carbon black
tape on an aluminium stub. A chromium sputter coating of 10 nm was carried out under vacuum with
a Cressington Sputter Metal 208 HR (Cressington, Watford, UK).
Catalysts 2017, 7, 54
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3.4.4. Nitrogen Physisorption
Adsorption–desorption isotherms of nitrogen were determined at 77 K using a Micromeritics
Tristar 3000 instrument (Micromeritics, Brussels, Belgium). Samples were degassed at 120 ◦ C under
dynamic vacuum for 24 h prior to textural measurements. Specific surface area were determined by
the Brunauer–Emmet–Teller Method (BET).
3.5. Transamination Activity Determination
A model reaction was used to assess catalytic activity of ATA-117: the transamination of pyruvate
with racemic BMBA, to produce BAP and D-alanine. As the enzyme only accepts R-BMBA as a
substrate, this reaction is a kinetic resolution. Typical conditions were 30 ◦ C, phosphate buffer 0.1 M
pH 8, pyridoxal phosphate 2.02 mM, sodium pyruvate 10 mM, racemic BMBA 10 mM, DMSO 5%.
Batch reactions were carried out in triplicates (plus a blank) in 5 mL round bottom glass flasks under
moderate magnetic stirring.
Transamination reaction conversion was determined on 100 µL sample taken from the reaction
medium. Ten microliters of sodium hydroxide (2 M) was added and the mixture was vortexed for 1 s.
Five hundred microliters of dichloromethane was then added to the aqueous phase and vortexed for
10 s to allow extraction of BAP and BMBA into the organic phase. Extraction step was repeated twice
and the organic phase was collected and analyzed by gas chromatography (analysis performed on
a Bruker Scion 456-GC with a WCOT fused silica BR-5 column (30 m × 0.32 mm ID × 1.0 µm) and
helium as carrier gas (25 mL/min), oven temperature at 150 ◦ C, split ratio of 20, injector temperature
at 250 ◦ C, flame ionization detector temperature at 300 ◦ C (air flow 300 mL/min, H2 flow 30 mL/min)
(Bruker MicroCT, Kontich, Belgium). BAP yield is defined as the proportion of rac-BMBA converted to
BAP (in %).
3.6. Enantiomeric Excess Determination
Enantiomeric excess of bromo-α-methylbenzylamine was determined by normal phase high
performance liquid chromatography (HPLC) (Hitachi-koki, Chiyoda, Japan) at 252 nm using a
Merck-Hitachi LaChrom L-7000 HPLC and a Chiralpak AD-H (250 mm × 4.6 mm) column with a
flow rate of 1 mL/min (90% isohexane/10% 2-propanol/0.1% diethylamine) for 10 min. The retention
times of the S- and R-enantiomers were 4.32 and 5.42 min, respectively. Specific rotation of the BMBA
product was established by comparison to known standards.
3.7. Enzyme Quantitation
Enzymatic concentrations were assessed by a modified Bradford method [37,38], using the Bovine
Serum Albumin protein as a standard. Five hundred microliters of sample were added to 1500 µL of
Bradford reagent. After 10 min incubation at room temperature, absorbances were read at 595 and
470 nm with a spectrophotometer ThermoScientific Genesys 10S-Vis (Thermo Fisher Scientific Inc.,
Waltham, MA, USA).
3.8. Dynamic Light Scattering (DLS)
DLS experiments were performed on a Malvern CGS-3 apparatus (Malvern Instruments, Hoeilaart,
Belgium) equipped with a He-Ne laser with a wavelength of 633 nm. The measurements were
performed at a 90◦ angle. Solutions were analyzed without any treatment. Five measurements were
taken for each sample (analysis duration was 30 s for each measurement). The data were analyzed
using the constrained regularization method for inverting data method, based on inverse-Laplace
transformation of the data.
Catalysts 2017, 7, 54
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3.9. Flow Reactions
3.9.1. Flow Reactor
A monolith was inserted into a heat-shrinkable PTFE tube, which perfectly fits the monolith
shape when heated preventing preferential flows along monolith sides. Heat-shrinkable PTFE tube
was also shrunk around Swagelok stainless steel male connectors (surrounded by PTFE tape to ensure
sealing), themselves connected to PTFE pipes. Flow device was connected to a PP Terumo syringe.
Syringe-pumps were used to deliver a controlled liquid flow. Monolithic reactors were placed in a
thermo-regulated water bath.
3.9.2. Transaminases Immobilization
Each reactor was impregnated with 50 mL of 0.2 g/L enzyme solution in buffer (potassium
phosphate buffer 0.1 M pH 8, pyridoxal phosphate 2.02 mM, sodium pyruvate 10 mM) with a flow
rate of 1.4 mL/min, with the water bath maintained at 6 ◦ C with ice packs. The effect of the enzyme
concentration for loading the monolith was investigated and 0.2 g/L was found to be sufficient to
reach maximal loading (Figure S10). Then, the reactor was washed at room temperature with 50 mL
buffer, with a flow rate of 1.4 mL/min, until no enzyme is detected in the outflow. Practically, this was
achieved after using approximately 30 mL of buffer solution. In some cases (covalent grafting), 50 mL
of a 1% glutaraldehyde aqueous solution was passed through the reactor (flow rate of 1.4 mL/min) at
room temperature before enzyme impregnation.
3.9.3. Flow Transamination Reaction
The model reaction was used to assess catalytic activity of ATA-117. Reaction medium (same
conditions as in batch reactions) was loaded in syringe and injected into the reactor with a flow rate
of 0.11 mL/min. Catalytic activity was determined by collecting the outflows and measuring BMBA
consumption and BAP production in gas chromatography. During enzymatic reaction, actual flow
rates were continuously monitored allowing an accurate determination of contact time. Porous volume
was estimated by weighing monolith before and after reaction (reaction medium density was measured
at 1.0024).
4. Conclusions
For the first time, ω-transaminases have been successfully immobilized on porous silica monoliths
and used in flow transamination reaction. When covalently grafted on APTES-functionalized silica
monoliths, using glutaraldehyde as a coupling agent, the enzyme shows good activity and full
enantioselectivity allowing the total kinetic resolution of racemic amines mixture. Biocatalysts were
stable both over time and upon storage. This study shows that such heterogeneous biocatalyst exhibits
all the required features for an enzymatic flow process. It paves the way to the development of
industrially relevant processes in continuous flow mode, based on heterogeneous biocatalysis, and in
particular the greener production of chiral amines.
Supplementary Materials: The following are available online at www.mdpi.com/2073-4344/7/2/54/s1,
Figure S1: Si(HIPE) monolith synthesis, Figure S2: Si(HIPE) monolith texture and SEM micrograph, Figure
S3: ATA-117 Protein sequence and surface properties, Figure S4: Loading estimation for TA-A50-GA samples,
Figure S5: Dynamic light scattering analysis of ATA-117 solutions, Figure S6: Kinetic resolution in batch mode
(yield and enantioselectivity), Figure S7: Flow reactor manufacture with heat-shrinkable membrane, Figure S8:
Plug flow regime into Si(HIPE) monolith, Figure S9: Non-enzymatic reactions (batch and flow), Figure S10:
Activity dependence on flow rate, Figure S11: Activity dependence on contact time.
Acknowledgments: The financial support of the Fond Spécial de Recherche from Université catholique de Louvain
is gratefully acknowledged. Authors also acknowledge the Communauté française de Belgique for the financial
support—including the PhD fellowship of L. van den Biggelaar—through the ARC programme (15/20-069).
Author Contributions: L.v.d.B. performed the experiments, analyzed the data and wrote the paper. P.S. and
D.P.D. conceived and designed the experiments, analyzed the data and wrote the paper.
Catalysts 2017, 7, 54
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Conflicts of Interest: The authors declare no conflict of interest.
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