Transaminase-Catalyzed Racemization with Potential for Dynamic Kinetic Resolutions

Article abstract of DOI:10.1002/cctc.201801049

Dynamic kinetic resolution (DKR) reactions in which a stereoselective enzyme and a racemization step are coupled in one pot would represent powerful tools for the production of enantiopure amines through enantioconvergence of racemates. The exploitation of DKR strategies is currently hampered by the lack of effective, enzyme-compatible and scalable racemization strategies for amines. In the present work, the proof of concept of a fully biocatalytic method for amine racemization is presented. Both enantiomers of the model compound 1-methyl-3-phenylpropylamine could be racemized in water and at room temperature using a couple of wild-type, non-proprietary, enantiocomplementary amine transaminases and a minimum amount of pyruvate/alanine as a co-substrate couple. The biocatalytic simultaneous parallel cascade reaction presented here poses itself as a customizable amine racemization system with potential for the chemical industry in competition with traditional transition-metal catalysis.

Full text of DOI:10.1002/cctc.201801049

Accepted Article
Title: Transaminase-catalyzed racemization with potential for dynamic
kinetic resolutions
Authors: Federica Ruggieri, Luuk M van Langen, Derek T Logan, Björn
Walse, and Per Berglund
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemCatChem 10.1002/cctc.201801049
Link to VoR: http://dx.doi.org/10.1002/cctc.201801049
A Journal of
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ChemCatChem
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FULL PAPER
Transaminase-catalyzed racemization with potential for dynamic
kinetic resolutions
[
a][b]
[c]
[b]
[b]
[a]
Federica Ruggieri,
Luuk M. van Langen, Derek T. Logan, Björn Walse, Per Berglund*
Abstract: Dynamic kinetic resolution (DKR) reactions in which a
stereoselective enzyme and a racemization step are coupled in one
pot would represent powerful tools for the production of enantiopure
amines through enantioconvergence of racemates. The exploitation
of DKR strategies is currently hampered by the lack of effective,
enzyme-compatible and scalable racemization strategies for amines.
In the present work, the proof of concept of a fully biocatalytic
method for amine racemization is presented. Both enantiomers of
the model compound 1-methyl-3-phenylpropylamine could be
racemized in water and at room temperature using a couple of wild-
type, non-proprietary, enantiocomplementary amine transaminases
and a minimum amount of pyruvate/alanine as a co-substrate
couple. The biocatalytic simultaneous parallel cascade reaction
presented here poses itself as a customizable amine racemization
system with potential for the chemical industry in competition with
traditional transition-metal catalysis.
these methods, DKR, in which a highly enantioselective enzyme
is combined in one pot with a compatible racemization system,
is of particular interest since it would allow the synthesis of
valuable enantiopure amines with yields close to 100% from
more readily accessible racemates.
The applicability of DKR to amines is limited by the scarcity of
[
7]
reported efficient enzyme-compatible racemization methods,
which usually require expensive transition-metal chemo-
[
7-9]
[8,10]
[11-15]
catalysts
(mostly Raney-Ni,
Shvo-type,
palladium-
[
10,15-19]
[20]
or ruthenium based ), expensive non-selective reducing
agents such as ammonia borane
[
21]
[3,22]
and/or harsh conditions
that are not suitable for large-scale application or in combination
with enzymatic steps. The best-known example of a successful
DKR on
a laboratory scale was reported by Reetz and
Schimossek in 1996. In this work, the substrate (R,S)-1-
phenylethylamine was successfully converted into its (R)-
acylated form to final 64% yield in 8 days using a lipase as
resolving catalyst and Pd on charcoal as racemization
[23]
catalyst. Extensive research on the combination of lipases and
heterogeneous racemization catalysts in DKRs has followed this
Introduction
[
24]
first report.
Enantiomers of a drug are known to exert different physiological
effects. Regulatory agencies have therefore progressively
tightened the requirements for the approval and the
Amine transaminases (EC 2.6.1.-) are biocatalysts of special
interest for the obtainment of chiral amines. This is not only
because both (R)- and (S)-selective amine transaminases with
[
1]
commercialization of chiral drugs, and this trend is likely to
continue in the future. For this reason, chiral amines used as
resolving agents, chiral auxiliaries or synthons in the synthesis
of pharmaceutical compounds are particularly interesting from
[25-
varying substrate scopes and operational optima are known.
26]
Also the reversibility of the reaction they catalyze, i.e. the
stereoselective transfer of an amino group from a chiral amino
donor substrate to a prochiral carbonyl amino acceptor co-
[2-3]
the commercial point of view.
Over the last 20 years, several
substrate, makes them suitable for both the kinetic resolution
biocatalytic routes for the synthesis of valuable chiral amines
have been investigated with the purpose of establishing more
environmentally friendly and affordable routes than the
traditional racemate resolution via formation of diastereoisomeric
[27]
and asymmetric synthesis of chiral amines.
Limitations such
as unfavorable thermodynamic equilibrium in asymmetric
[6,28]
synthesis and substrate- and product inhibition phenomena
[3,27,29-35]
have been often overcome by reaction-
and/or enzyme
[
3-5]
salts.
Several biocatalytic approaches for the synthesis of
[27,29]
engineering approaches.
enantiopure chiral amines have also been reported, mostly
involving hydrolases, oxygenases, dehydrogenases and
transferases in kinetic resolution (KR), dynamic kinetic resolution
One of the most interesting applications of transaminases in
biocatalysis consists of a racemization reaction intended for use
in DKRs. Koszelewski et al. designed a one-pot cascade
involving a pair of known, robust enantiocomplementary amine
transaminases coupled via catalytic amounts of a non-readily-
available and rather expensive achiral amino donor/acceptor co-
[
3-6]
(
DKR) and asymmetric synthesis (AS) reactions.
Among
[
a]
F. Ruggieri, Prof. P. Berglundꢀ
Department of Industrial Biotechnologyꢀ
KTH Royal Institute of Technologyꢀ
[36]
substrate system.
In this approach, the selection of
AlbaNova University Center, SE-106 91 Stockholm (Sweden)ꢀ
E-mail: per.berglund@biotech.kth.se
appropriate pairs of catalysts is complicated by the natural
narrow substrate specificity overlap between (R)- and (S)-
transaminases and by the need to use accepted achiral co-
substrates that must also both be commercially available and
show a readily reversible transamination equilibrium with the
substrate in order to be used in catalytic amounts. The difficulty
to meet all these requirements at once has limited further
practical applications of the system.
[
[
b]
c]
F. Ruggieri, Dr. D. T. Logan, Dr. B. Walseꢀ
SARomics Biostructures AB,ꢀ Medicon Village,ꢀ Scheelevägen 2,
SE-223 81 Lund (Sweden)
Dr. L. M. van Langen
Viazym BV
Molengraaffsingel 10, 2629JD Delft (The Netherlands)
Supporting information for this article is given via a link at the end of
the document.
In the present paper, the proof of concept of a more accessible
transaminase-catalysed racemization reaction is presented, in
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ChemCatChem
10.1002/cctc.201801049
FULL PAPER
which the use of the broadly accepted alanine/pyruvate co- Results and Discussion
substrate system increases the chances to identify a couple of
non-proprietary enantiocomplementary catalysts and limits the
goal of engineering, when necessary, to only one of the two
substrates participating in the reaction. Our results show that
both enantiomers of the model compound 1-methyl-3-
phenylpropylamine (1a,b) could be racemized to a final <10%
e.e. using two non-proprietary enantiocomplementary amine
transaminases selected from a panel of only 10 enzymes (7 (R)-
selective and 3 (S)-selective) and using a variant of the simple
and inexpensive UV-based acetophenone assay. Purified
enzymes (50 mU) were used in water, at pH 7.5, at atmospheric
pressure, in the presence of the enantiopure substrate 1a, (S)-
enantiomer, or 1b, (R)-enantiomer, (15 mM) and minimal
amounts of enantiopure alanine of opposite configuration than
that of the target amine (3a, L-alanine, or 3b, D-alanine)
Selection and production of catalysts and choice of the
operational pH
Suitable catalysts were selected among a collection of enzymes
including those commercially offered in the ACS-ATA-KIT
(
(
Enzymicals AG, Table S1) and the Chromobacterium violaceum
[
37]
[38]
S)-ATA [Cv-(S)-ATA] in its wild-type
and W60C variants.
The enzymes were screened for the acceptance of ketone 2 in
[
39]
the variant of the acetophenone assay
shown in Figure S1.
The Aspergillus oryzae (R)-ATA [Ao-(R)-ATA] and the Cv-(S)-
ATA showed the highest initial catalytic rates and were therefore
selected for use in the racemization reaction, which was
[
26,40]
performed at pH 7.5 to allow for sufficient enzyme activity
[
41]
and better (S)-ATA stability. Both the Ao-(R)-ATA and the Cv-
S)-ATA were then recombinantly expressed in E. coli
(
(
Scheme 1). In this setup the catalytic activity of the two
BL21(DE3) and used in the racemization of 1a and 1b in their
pure form to avoid the occurrence of side eactions and to have a
tight control over the relative amounts of the two catalysts, which
is a key paramenter in the early-stage optimization of any multi-
enzymatic reaction. While the Cv-(S)-ATA was expressed and
enzymes is coupled via continuous production and consumption
of the amino acceptor pyruvate 4, which is added to the reaction
in limited amounts. Although distant from the metrics necessary
for industrial application, the approach presented here
represents a promising novel, tunable and optimizable enzyme-
compatible racemization system for amines with potential for
DKR processes.
[
42]
purified from the strain previously described in the literature
following established procedures,
[38,42]
the catalytically active C-
terminal His-tagged Ao-(R)-ATA was successfully over-
expressed (Figures S3 and S4, Supporting Information) and
purified by IMAC for the first time with yields around 70 mg/L.
Removal of imidazole resulted in fast and irreversible protein
precipitation, suggesting a stabilizing role of imidazole for Ao-
(
R)-ATA. Flash-freezing in liquid nitrogen and storage at -80 °C
proved optimal for the long-term storage of both enzymes as it
prevented loss of activity even over repeated cycles of freeze-
[
43]
thawing. The concentration of catalytically active protein
in
the final enzyme preparations was assessed using a standard
UV-based activity assay in the presence of a known amount of
[
39]
purified protein.
The calculated specific activity values,
corresponding to 103 mU/mg for Cv-(S)-ATA and 169 mU/mg for
Ao-(R)-ATA (Figure S1, Supporting Information), account for the
presence of inactive forms of the enzymes in the protein
preparation and enable a tight control over the actual amount of
catalysts added to the reaction.
Transaminase-catalyzed racemization of 1a and 1b
Racemization of the model amine compounds 1a and 1b was
achieved employing Cv-(S)-ATA and Ao-(R)-ATA (50 mU each)
in a one-step reaction in the absence of excess amino donor or
additional equilibrium-displacement systems (Scheme 1). The
two enantiomers 1a and 1b of the target 1-methyl-3-
phenylpropylamine (15 mM) were independently racemized in
aqueous solution in the presence of minimal amounts of amino
acceptor pyruvate 4 (4.5 mM, 0.3 eq.), amino donor alanine 3a
or 3b (10 mM, 0.7 eq.) and PLP (0.1 mM) over a reaction time of
Scheme 1. Investigated racemizations of 1a (A) and 1b (B) (15 mM). The
reactions were performed using purified Cv-(S)-ATA and Ao-(R)-ATA (50 mU
each) as catalysts in presence of limiting amounts of amino acceptor 4 (4.5
mM) and amino donor 3b (A) or 3a (B) (10 mM) in HEPES pH 7.5 (50 mM),
glycerol (20% v/v) and PLP (0.1 mM) at room temperature in the dark for
four days.
4
days, which corresponds to the chemocatalyzed racemization
[3,17]
time for this compound reported in previous work.
The
chosen quantities ensure that racemization can only be the
outcome of the catalytic coupling of the two transaminases,
since only the enzyme-catalyzed replenishment of the otherwise
limiting amino-acceptor pyruvate 4 (0.3 eq.) can sustain the
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ChemCatChem
10.1002/cctc.201801049
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racemization cycle. For each reaction mixture, opposite
enantiomers of 1-methyl-3-phenylpropylamine (1a,b) and
alanine (3a,b) were used as substrate and amino donor co-
substrate, respectively. Although the addition of an enantiopure
amino donor cannot be avoided due to the strict
enantioselectivities of the enzymes, our results show that, in
contrast to other reported transaminase-based systems, only a
minimal amount of amino donor (0.67 eq.) suffices to sustain the
asymmetric synthesis half-reaction, with a positive impact on the
overall atom economy of the racemization reaction (Table 1).
The obtained data demonstrate that racemization of 1a,b could
be achieved to final ee <10% in the described bi-enzymatic
reaction, regardless of the starting enantiomer of 1, with
Table 1. Transaminase-catalyzed one-step racemization of 1a and 1b under
testedconditions.
Absolute
concentration of
Total
amount of
PLP
ee of
Starting
amine
Catalytic
system
ketone after 4
[
a]
amine after
[c]
[b]
days
4 days
[
mol (mol%)]
[% (mol)]
1
a
a
No enzymes
1.7 (11)
1.7 (11)
1.7 (11)
1.7 (11)
>99% (S)
9% (S)
3 (0.5)
36 (5.4)
4 (0.6)
Ao-(R)-ATA
Cv-(S)-ATA
1
1
b
b
No enzymes
>99% (R)
5% (R)
Ao-(R)-ATA
Cv-(S)-ATA
1
42 (6.3)
4 (0.6)
absolute concentrations of ketone
2 (4-phenyl-2-butanone)
[
d]
above 20% of the initial substrate concentration (>3 mM). The
presence of trapped intermediate ketone, i.e. ketone
accumulated during the course of the reaction and not further
converted to its corresponding amine, is inherent to this type of
1b
No enzymes
0.10 (0.7)
0.10 (0.7)
>99% (R)
3.8% (R)
[
d]
Ao-(R)-ATA
Cv-(S)-ATA
1b
28 (4.2)
[
3
a] Substrate amine 1a or 1b (15 mM), amino donor of opposite chirality 3b or
a (10 mM) and amino acceptor 4 (4.5 mM) in HEPES pH 7.5 (50 mM)
[
44]
racemization reaction
to an extent that depends on the
position of the equilibrium. The coupling to a kinetic resolution
step in a dynamic kinetic resolution system is expected to
contribute to a reduction of the amount of trapped ketone.
supplemented with PLP (0.1 mM). PLP (0.4 mol) was supplemented daily
unless otherwise specified. Equal amounts of catalysts (50 mU) was used,
when present. [b]Values obtained from chiral HPLC analysis. [c] Values
obtained from quantitative achiral GC. [d] PLP was not supplemented daily
after the start of the reaction.
PLP (0.4 μmol) was supplemented daily to the reaction to
ensure continuous cofactor availability. This cofactor addition
pattern proved both unnecessary for the purpose of
racemization and undesirable with respect to both product ee
and amount of trapped ketone intermediate. The control reaction
including substrate 1b supplemented with a minimal amount of
PLP only at time 0 (0.6 μmol) shows both better ee value and a
lower concentration of ketone. We suspect that PLP could
interfere stoichiometrically in the reaction by removing amino
groups from the system in the form of PMP and that this
phenomenon might be relevant for the accumulation of ketone
when excess PLP is used in combination with enzymes that, like
1a or 1b as substrates show similar progress curves, with a
slower increase of the relative concentrations of the two
enantiomers as the racemization equilibrium is approached. The
initial time points (0.5, 1 and 2.5 hours) describe the systems far
from racemization equilibrium, i.e. when one of the two amine
enantiomers is present in large excess. This situation resembles
the one encountered in dynamic kinetic resolutions, in which the
kinetic resolution step selectively and continuously removes one
of the enantiomers from the equilibrium. The 24-hours sample,
on the other hand, can provide some indication about long-term
differences in the two thermodynamically identical racemization
reactions, which can be assumed to arise exclusively from
events that alter the catalytic activity of the systems. By virtue of
the enantioselectivity of the catalysts involved, different
structural stabilities of the enzymes, sensitivities to inhibition
phenomena and propensities to cofactor leakage will affect the
racemization reactions differently depending on the chirality of
the starting (excess) enantiomer. In this case the concentration
ratio between the two enantiomers after 24 hours of reaction is
significantly different and we think that this might be due to a
combination of the aforementioned events. This phenomenon
was nonetheless not investigated further since for the intended
application of the reaction under study, i.e. DKR, the behaviour
of the system far from the racemization equilibrium and in
presence of sufficiently stable enzymes is of interest. The
racemization times (trac ) of the systems, herein defined as the
time that would be required to achieve complete racemization of
an enantiopure substrate when the initial catalytic rate is held
constant, correspond to 13.6 hours for substrate 1b and 19.8
hours for substrate 1a (see Supplementary Information). This
value could be further reduced by increasing the amount of
catalysts. The racemization time trac also represents the time that
would be required to complete a DKR reaction when the kinetic
[43]
Cv-(S)-ATA, show low affinity for the cofactor moiety.
Based on the presented results, we believe that the described
approach should be further investigated for different types of
substrate amines for DKR purposes. Appropriate system- and
substrate-dependent optimizations are expected to be needed.
The attractiveness of a fully enzyme-compatible and renewable
system for racemization of primary amines justifies, in the
context of what is currently available, further efforts in this
direction.
Racemization kinetics
The success of dynamic kinetic resolution (DKR) reactions
strictly depends on both the selectivity of the enzyme catalyzing
the kinetic resolution step and the racemization rate of the slow-
reacting enantiomer, which should ideally be at least ten-fold
[
22,24]
faster than the kinetic resolution step.
The kinetics of the
transaminase-catalyzed racemization of 1a and 1b were
investigated by monitoring the change in the relative
concentrations of the two enantiomers over time (Figure 1) in
reaction setups identical to those already described in which
PLP (0.1 mM) was exclusively added at time 0. Samples of the
reactions were collected and immediately derivatized for
analysis at 0.5, 1, 2.5 and 24 hours. Both reactions using either
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ChemCatChem
10.1002/cctc.201801049
FULL PAPER
n-heptane (Fisher Scientific) and anhydrous sodium sulfate (Merck). All
solvents used were of analytical grade. Pyruvate and pyridoxal-5’-
phosphate were also purchased from Sigma Aldrich, as sodium salt and
monohydrate form, respectively.
For protein purification procedures syringe filters (0.45 and 0.22 µm)
were purchased from Corning, centrifugal filter units (Amicon Ultra-15, 30
kDa and 50 kDa) from Merck Millipore, Ni-NTA affinity chromatography
pre-packed cartridges for affinity chromatography (5 ml) from Nordic
Biosite and Sephadex G-25 PD-10 desalting columns (2.5 ml) from GE-
Healthcare.
Catalyst selection
Catalysts were selected among the panel of enzymes commercially
offered by Enzymicals AG in the ACS-ATA-KIT and those kindly made
available by the KTH biocatalysis group. The variant of the acetophenone
[
39]
assay shown in Figure S1 aided the identification of suitable candidate
catalysts by direct comparison of the detected initial catalytic rates.
Enzyme solutions of all available catalysts (1 mg/ml, whole-cell
lyophilizate or purified lyophilized enzyme) were prepared in HEPES pH
7.5 (50 mM) supplemented with PLP (0.1 mM). The substrate solution
was prepared dissolving the amino acceptor 4-phenyl-2-butanone (5 mM)
and the acceptor methylbenzylamine (5 mM) (either as (R)- or (S)-
enantiomer) in HEPES pH 7.5 (50 mM). The assay solutions were
prepared mixing buffer (495 μl), enzyme solution (5 µl) and substrate
solution (500 μl) in clean quartz cuvettes. Formation of acetophenone
was monitored following the increase of A245nm over 10 minutes. Initial
activities were estimated from the linear regression of the first data
points. The enzymes showing highest initial catalytic rates (Aspergillus
oryzae (R)-ATA and Chromobaterium violaceum (S)-ATA) were selected
for further investigations.
Figure 1. Reaction course for the racemization reactions performed using
enantiopure 1a or 1b (15 mM) as substrates in presence of the opposite
alanine enantiomer 3b or 3a (10 mM), amino acceptor 4 (4.5 mM) and purified
catalysts (50 mU each). The reactions (1 ml) were run in HEPES pH 7.5 (50
mM) at room temperature and in the dark under mild agitation. Cofactor PLP
(0.1 mM) was only added at the beginning of the reaction. *Data referring to
the first experiment where fresh PLP (0.4 μmol) was added every 24 hours.
resolution step is not the rate-limiting reaction and the
racemization proceeds at its fastest rate.
Conclusions
Design of expression plasmids and production of catalysts-producing
bacterial strains
In the present paper a novel biocatalytic method for the
racemization of primary amines is presented that relies on the
use of two wild-type, non-proprietary, enantiocomplementary
amine transaminases and that is sustained by internal formation
and consumption of the amino acceptor pyruvate. The system
enabled the one-pot racemization of the model amine substrate
Aspergillus oryzae (R)-ATA (Ao-(R)-ATA)
The Ao-(R)-ATA coding sequence was retrieved from the results of a
default BLAST search in which the Aspergillus fumigatus (R)-ATA
sequence (UniProt KB ID: Q4WNL4) was used as query. Two sequences
from Aspergillus oryzae were retrieved, both sharing 73% identity with
the query and differing by only 4 amino acid residues (L/V 7, M/I 53, A/V
1-methyl-3-phenylpropylamine starting from either one of its
enantiomers and using only minimal amounts of enantiopure D-
or L-alanine as amino-donor co-substrate. The system, which
shows reduced complexity and better atom economy when
compared to previous reports, represents a potentially flexible
racemization tool, optimizable to meet substrate- and process
requirements. We believe that the described racemization
system, being it performed under mild reaction conditions that
well match the requirements of other biocatalysts, could be
exploited in DKR reactions for deracemization applications.
248 and K/E 293). The variant LMAK (GenBank: EIT82926.1) was
reverse-translated using the tool available on
www.bioinformatics.org/sms2. The nucleotide sequence was then codon-
optimised for E.coli and supplemented with NdeI and XhoI terminal
restriction sites. Gene synthesis, restriction and ligation in pET-29b were
®
purchased from GenScript . The final Ao-(R)-ATA construct included a
C-terminal LE insertion between the enzyme sequence and the
hexahistidine tag. The plasmid was transformed into electrocompetent E.
coli BL21(DE3) cells using a Bio-Rad Gene Pulser II. A single colony
growing on LB agar plates supplemented with kanamycin was inoculated
into sterile liquid LB medium (15 ml) supplemented with kanamycin (50
mg/L) and incubated overnight at 37 °C and 200 rpm agitation. This
preculture (5 ml) was subsequently inoculated in sterile LB medium (50
ml) supplemented with antibiotic. Growth was carried out at 37 °C and
200 rpm agitation until OD600 = 0.56. Glycerol stocks were prepared in
Experimental Section
Materials
40% v/v glycerol, frozen in liquid nitrogen and kept at -80 °C for long term
All chemicals were purchased from Sigma Aldrich at analytical grade with
the exceptions of HEPES (buffer grade), D-alanine (Fluka), imidazole
storage.
(buffer grade), (R)- and (S)-1-methyl-3-phenylpropylamine (ACROS
Organics), isopropyl-ether (stabilized with BHT, TCI), 2-propanol (Roth),
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ChemCatChem
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FULL PAPER
Chromobacterium violaceum (S)-ATA (Cv-(S)-ATA)
(cut-off 50 kDa). The imidazole was removed by desalting using a PD-10
column (GE Healthcare). After a final concentration step to 37.5 mg/ml
the protein was flash frozen and stored at -80 °C.
The E.coli BL21(DE3) expressing strain was made available by the KTH
[
42]
biocatalysis group in the form of a glycerol stock.
expression vector carried the Chromobacterium violaceum (S)-TA (Cv-
S)-TA) coding sequence in fusion with an N-terminal hexahistidine tag.
The pET28a
Activity estimation
(
Enzyme activity was estimated in the variant of the acetophenone assay
shown in Figure S1 (Supporting Information) using UV-clear disposable
cuvettes and a Cary 3000 spectrophotometer. The final assay solution
was obtained mixing 1 ml substrate solution (5 mM 4-phenyl-2-butanone,
Heterologous expression of catalysts
Aspergillus oryzae (R)-ATA
5
mM (R)- or (S)-methylbenzylamine) and 50 μg purified enzyme, added
as 5 μl of a 10 mg/ml enzyme dilution prepared from the frozen stocks,
whose real enzyme content was estimated on NanoDrop
spectrophotometer (ThermoFisher Scientific) using the molar extinction
Liquid sterile LB medium (15 ml) supplemented with kanamycin (50
mg/L) was inoculated with cells from the glycerol stock. After overnight
growth at 37 °C and 200 rpm agitation the preculture (1 ml) was used as
inoculum in sterile TB medium (1 L) supplemented with kanamycin (50
mg/L). Cultivation was performed in 5-liter baffled flasks at 37 °C and 180
rpm agitation. Cells were induced with IPTG (0.3 mM) at values of
OD600 ranging from 0.6 to 0.8. Expression was carried out for 20 hours
at 20 °C in the dark. Cells were harvested by centrifugation (4000 g for
a
[45]
coefficients calculated in ProtParam (E 0.1% = 1.560 and 1.239 for Cv-
245nm
S)-ATA and Ao-(R)-ATA, respectively). The increase of A ,
(
corresponding to the absorption maximum of the ketone product
acetophenone, was monitored over an assay time of three minutes. The
averaged values of the slopes obtained from the assay performed in
triplicate were taken as reliable values for the initial enzymatic rates.
From these values, using the molar extinction coefficient of
acetophenone (ε = 12 mM cm ), the enzyme concentration could be
expressed in [mU/mg] (103 mU/mg for Cv-(S)-ATA and 169 mU/mg for
Ao-(R)-ATA).
20 minutes at 4 °C) and the resulting pellets were stored at -20 °C until
purification.
[
46]
-1
-1
Chromobacterium violaceum (S)-ATA
All steps of cultivation, induction, harvest and storage were performed
following the same protocol optimized for Ao-(R)-RATA.
Racemization reaction setup
All reactions and controls (1 ml) were performed in HEPES pH 7.5 (50
mM) supplemented with glycerol (20% v/v) in sealed 1.5 ml glass vials
kept in the dark under mild agitation at room temperature for 4 days.
The racemization of the model compound 1-methyl-3-phenylpropylamine
Purification of catalysts
Aspergillus oryzae (R)-ATA
(15 mM) was tested starting from each one of its pure enantiomers 1a
The Ao-(R)-ATA was purified by IMAC. Cell pellets were resuspended
and lysed on ice in 50 mM HEPES pH 7.5, 0.2 M NaCl, 5 % v/v glycerol,
and 1b (purchased from ACROS organics) in the presence of the
opposite alanine enantiomer 3b or 3a (10 mM) as amino donor for the
asymmetric synthesis half-reaction and pyruvate 4 (4.5 mM) as amino
acceptor in the kinetic resolution half-reaction (Figure 1). PLP (0.1 mM)
was also added to reactions and controls at time zero. Additional cofactor
(0.4 µmol) was supplemented daily to all mixtures with the exception of
one control racemization reaction (containing 1b as substrate) in which
no additional PLP was added after time zero. The reactions were started
upon simultaneous addition of Ao-(R)-ATA and Cv-(S)-ATA (50 mU
each) to the reaction mixtures. The corresponding volume in buffer was
added to each control mixture.
1
0 mM imidazole using a Branson Sonifier 250 equipped with a 3 mm
probe. Three sonication cycles of seven minutes each were performed
40% duty cycle, output level 4.5). After sedimentation of the cell debris
(
by centrifugation at 40000 g for 30 minutes at 4 °C the soluble fraction
was further filtered (cut-off 0.22 µm) to ensure complete removal of
particulate matter. The resulting sample was loaded onto
a pre-
equilibrated 5 ml Ni-NTA column (Nordic Biosite) and washed at a flow
rate of 2 ml/min with increasing concentrations of imidazole (10 mM, 50
mM, 250 mM, three column volumes for each concentration). A linear
gradient to final 500 mM imidazole was then used to elute the target
(duration of the gradient 30 minutes, flow rate 2 ml/min). The PLP-
Reaction workup and analytics
saturated protein eluted as single symmetric peak at 290 mM
a
imidazole. Attempts to remove the imidazole resulted in protein
precipitation. The protein was therefore concentrated to 15 mg/ml in 50
mM HEPES pH 7.5, 0.2 M NaCl, 5 % v/v glycerol, 290 mM imidazole
using Amicon Ultra centrifugal filter units (cut-off 30 kDa), flash frozen
and stored at -80 °C.
Samples for analysis (200 µl) were transferred to clean tubes and
basified adding 1 M NaOH (20 µl). Ethyl acetate (220 µl) was then used
to perform extraction. Phase separation was achieved by centrifugation.
GC quantitative analysis of 4-phenyl-2-butanone
Chromobacterium violaceum (S)-ATA
GC quantitative analysis of 4-phenyl-2-butanone was performed on a
Varian Star 3400 CX equipped with an Agilent DB-5ms Ultra Inert column
Cell pellets were resuspended and lysed in 50 mM HEPES pH 7.5, 10
mM imidazole following the same protocol described for Ao-(R)-ATA.
Clarification and loading onto the affinity column (Nordic Biosite, 5 ml)
also followed the same procedures reported for Ao-(R)-ATA. After
washing with five column volumes of buffer, the target was eluted in a
linear gradient to 500 mM imidazole in 30 minutes, flow rate 2 ml/min.
The protein eluted in a single symmetric peak at 350 mM imidazole. The
fractions corresponding to the peak were collected, merged and
concentrated by ultrafiltration using Amicon Ultra centrifugal filter units
(
2
30 m x 0.25 mm x 0.25 m) and a FID detector using N as carrier.
2
A calibration curve for the target 4-phenyl-2-butanone (R = 98.5%) was
obtained in the concentration range 2-8 mM using acetophenone (15 mM
in anhydrous ethyl acetate) as external standard. For the sample
analysis, the acetate phase obtained from the extraction (90 μl) was dried
over anhydrous sodium sulfate and a small amount (38 μl) was mixed
with the external standard (10 μl) in a clean tube. After 1:2 dilution in
anhydrous ethyl acetate, 1 μl of sample was manually injected for
analysis. The GC method consisted in a linear temperature gradient (10
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ChemCatChem
10.1002/cctc.201801049
FULL PAPER
°
9
C/min) from 50 °C (initial time 2 min) to 250 °C. Retention times were
.5 min for the external standard acetophenone and 11.79 min for the
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HPLC qualitative analysis of N-acetyl-(R,S)-1-methyl-3-
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3
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(
R)- and (S)-enantiomers, respectively. In the case of the samples used
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The authors gratefully acknowledge funding from the European
Commission under the Horizon 2020 program through the Marie
Skłodowska-Curie action BIOCASCADES ITN-EID (grant
agreement 634200). All BIOCASCADES consortium members
are also sincerely acknowledged for scientific discussion and
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Keywords: Chiral amines • Enzyme catalysis • Racemization•
Transaminase
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Dynamic kinetic resolution
Federica Ruggieri, Luuk M. van Langen,
Derek T. Logan, Björn Walse, Per
Berglund*
approaches for chiral primary amines
are hindered by the scarcity of cheap,
renewable and enzyme-compatible
racemization strategies. We report
here a fully biocatalytic one-step
reaction for the reacemization of chiral
primary amines. Two
Page No. – Page No.
Transaminase-catalyzed racemization
with potential for dynamic kinetic
resolutions
enantiocomplementary amine
ransaminases are employed in mild
conditions and in absence of excess
co-substrates or auxiliary equilibrium
displacement systems, the reaction
being solely sustained by the internal
recycling of the natural amino
acceptor pyruvate. The system
represents an optimizable setup for
implementation in DKRs.
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Title
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