Isopropylamine as Amine Donor in Transaminase-Catalyzed Reactions: Better Acceptance through Reaction and Enzyme Engineering

Article abstract of DOI:10.1002/cctc.201800936

Amine transaminases (ATA) have now become frequently used biocatalysts in chemo-enzymatic syntheses including industrial applications. They catalyze the transfer of an amine group from a donor to an acceptor leading to an amine product with high enantiopurity. Hence, they represent an environmentally benign alternative for waste intensive chemical amine synthesis. Isopropylamine (IPA) is probably one of the most favored amine donors since it is cheap and achiral, but nevertheless there is no consistency in literature concerning reaction conditions when IPA is best to be used. At the same time there is still a poor understanding which structural properties in ATA are responsible for IPA acceptance. Herein, we demonstrate, on the basis of the 3FCR enzyme scaffold, a substantial improvement in catalytic activity towards IPA as the amine donor. The asymmetric synthesis of industrial relevant amines was used as model reaction. A systematic investigation of the pH-value as well as concentration effects using common benchmark substrates and several ATA indicates the necessity of a substrate- and ATA-dependent reaction engineering.

Full text of DOI:10.1002/cctc.201800936

Accepted Article
Title: Isopropylamine as Amine Donor in Transaminase-Catalyzed
Reactions: Better Acceptance through Reaction and Enzyme
Engineering
Authors: Ayad Dawood, Martin Weiß, Christian Schulz, Ioannis
Pavlidis, Hans Iding, Rodrigo de Souza, and Uwe
Bornscheuer
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.201800936
Link to VoR: http://dx.doi.org/10.1002/cctc.201800936
A Journal of
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ChemCatChem
10.1002/cctc.201800936
COMMUNICATION
natural substrate and especially the position of the transferred
[
1,2]
amine group and/or carboxyl moiety.
Amine transaminases
Isopropylamine as Amine
Donor in Transaminase-
Catalyzed Reactions: Better
Acceptance through Reaction
and Enzyme Engineering
(
ATA), a subgroup of ω-TA (class III transaminase family), are of
special interest for the chemo-enzymatic application. In contrast
to ω-TA, which accept carbonylic substrates with a distal
carboxylate group, ATA tolerate substrates without a carboxyl
moiety and therefore a wide range of ketones and aldehydes.
Hence, in the last decade ATA became very attractive targets for
[
3–13]
enzyme engineering
alternative for the chemical transition metal-catalyzed amine
representing an environmentally benign
[
14]
synthesis in pharmaceutical and agrochemical industries. For
[
10]
[15,16]
instance, the production of imagabaline,
(S)-rivastigmine
[
a]
[a]
[17]
Ayad W.H. Dawood, Martin S. Weiß , Christian
or (S)-ivabradine has been realized on larger scale using ATA.
Certainly, one of the most notable examples is the
manufacturing of (R)-sitagliptin in >200 g/L scale after several
[
a]
[a,b]
[c]
Schulz , Ioannis V. Pavlidis , Hans Iding ,
[
d]
[
3]
Rodrigo O.M.A. de Souza, Uwe T.
rounds of protein engineering of the selected ATA. In this
specific example, enzyme engineering resulted not only in a
better substrate acceptance or higher temperature and solvent
stability, but also in an enhanced isopropylamine (IPA)
acceptance. The best variant contained 27 mutations. To have
the enzyme accepting the bulky prositagliptin ketone, the most
mutations were around the active site region. However, to
ensure acceptance/tolerance of IPA many mutations were
globally distributed over the entire protein since the majority of
wild-type ATA do not accept it well as the amine donor. More
recently, we could also design (S)-selective ATAs for the
asymmetric synthesis of chiral amines from sterically demanding
[
a]
Bornscheuer*
Abstract: Amine transaminases (ATA) have now become frequently
used biocatalysts in chemo-enzymatic syntheses including industrial
applications. They catalyze the transfer of an amine group from a
donor to an acceptor leading to an amine product with high
enantiopurity. Hence, they represent an environmentally benign
alternative for waste intensive chemical amine synthesis.
Isopropylamine (IPA) is probably one of the most favored amine
donors since it is cheap and achiral, but nevertheless there is no
consistency in literature concerning reaction conditions when IPA is
best to be used. At the same time there is still a poor understanding
which structural properties in ATA are responsible for IPA
acceptance. Herein, we demonstrate, on the basis of the 3FCR
enzyme scaffold, a substantial improvement in catalytic activity
towards IPA as the amine donor. The asymmetric synthesis of
industrial relevant amines was used as model reaction. A systematic
investigation of the pH-value as well as concentration effects using
common benchmark substrates and several ATA indicates the
necessity of a substrate- and ATA-dependent reaction engineering.
[
6,13,18]
bulky ketones.
The asymmetric synthesis is the most convenient and
economically favored route to a target chiral amine. In contrast
to the kinetic resolution mode, the asymmetric synthesis starts
from the prochiral ketone and results in the desired chiral
[
19–21]
product with a theoretical yield of 100%.
The downside of
this strategy is a very often unfavorable reaction equilibrium,
which makes strategies for equilibrium shifting necessary.
Therefore several equilibrium displacement techniques were
established, for instance involving enzymatic cascades in order
[
19,22–26]
Transaminases are versatile and widely used biocatalysts for the
production of chiral amines. They catalyze the transfer of an
amine group from an amine donor to a ketone or aldehyde
to remove co-products,
utilization of ‘smart-donors’ which
sacrificial co-substrates after
or application of the amine donor in large
are
converted
into
[
27–32]
transamination
[
33]
acceptor, following
a
Ping-Pong Bi-Bi-reaction mechanism
excess. In fact, IPA is the industrially favored amine donor for
asymmetric syntheses since it is cheap, achiral – so the
enantioselectivity of the ATA has not to be considered – and the
utilizing pyridoxal-5’-phosphate (PLP) as cofactor, revealing the
amine product in a high enantiopurity. Transaminases belong to
the fold types I and IV of PLP-dependent enzymes and are
additionally divided into six subclasses, depending on the
by-product acetone is supposed to have
a drastic lower
[
33]
reactivity in the back reaction. Additionally, in terms of shifting
the equilibrium, acetone can be easily removed from the
[
3]
reaction solution. In general, it was reported in many cases
that an excess of IPA needs to be applied to drive the
transamination of various substrates to the desired product
[
a]
M.Sc. Biochem. A.W.H. Dawood, Dr. M.S. Weiß, M.Sc. Biol. C.
Schulz, Assist. Prof. I.V. Pavlidis, Prof. Dr. U.T. Bornscheuer
Dept. of Biotechnology & Enzyme Catalysis
Institute of Biochemistry, Greifswald University,
Felix-Hausdorff-Str. 4
[
8,13,18,23,34–44]
side
when enzyme engineering did not lead to a
better IPA acceptance, as mentioned above. In particular,
different reaction conditions were reported such as varying
17487 Greifswald (Germany)
E-mail: uwe.bornscheuer@uni-greifswald.de
Web: http://biotech.uni-greifswald.de
Assist. Prof. I. V. Pavlidis Dept. of Chemistry, University of Crete,
Voutes University Campus, 70013 Heraklion, (Greece)
Dr. H. Iding Process Chemistry and Catalysis, Biocatalysis F.
Hoffmann–La Roche Ltd. Grenzacher Strasse 124, 4070 Basel
[
39]
[38]
[8]
donor-acceptor-ratio from 1.5-fold over 40-fold to 200-fold
[
41]
[45]
[
b]
c]
and a pH range from 7.3 to 9.5.
[
(
Switzerland)
[d]
Prof. Dr. R.O.M.A. de Souza
Biocatalysis and Organic Synthesis Group, Institute of Chemistry
Federal University of Rio de Janeiro (Brazil)
Supporting information for this article is given via a link at the end of
the document.
Scheme 1. Model reactions and preparative scale reactions with IPA as amine
donor using substrates as reported in literature.
[
6]
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ChemCatChem
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COMMUNICATION
Table 1. Comparison of the presented variants of 3FCR regarding initial activity measurements with racemic 1b or 2b and asymmetric synthesis of 1b or 2b
using IPA as the amine donor. * n.d.: not detectable
-
1
[a]
[b]
ATA variant
Specific activity [mU mg ]
Substrate conversion [%]
[
c]
[c]
rac-1b
rac-2b
1a
ee
P
[%]
2a
ee
P
[%]
-
3
3
3
3
FCR-QM
218.6 ± 16.8
88.1 ± 4.2
141.2 ± 1.6
70.9 ± 0.7
104.9 ± 1.6
70.3 ± 1.5
63.5 ± 0.6
90.9 ± 4.3
12.8 ± 1.2
6.8 ± 1.5
50.6 ± 3.6
83.5 ± 4.0
> 99 (R)
86 (R)
91 (R)
80 (R)
n.d.*
FCR-QM-W59L
8.9 ± 1.8
23.8 ± 2.5
85.8 ± 3.5
> 99 (S)
> 99 (S)
> 99 (S)
FCR-QM-W59L-R420A
FCR-QM-W59L-R420W
[
a] Assay conditions for the kinetic resolution: 0.5 mM rac-1b or 0.25 mM rac-2b, pentanal in equimolar ratio, 10% (v/v) DMSO, CHES pH 10 (50 mM), 0.045 –
-
1
0
.07 mg mL purified enzyme, 30 °C.
[
b] Assay conditions for the asymmetric synthesis: 2 mM ketone (1a or 2a), 0.5 M IPA, 30% (v/v) DMSO, CHES buffer pH 10 (50 mM), 0.1 mM PLP, 0.89 - 1.2 mg
-1
mL purified enzyme (substrate to enzyme ratio, s/e ~ 0.35 – 0.47 w/w), 30 °C. Conversion was determined after 20 h via gas chromatography (GC) with
-iodoacetophenone as internal standard for the quantification of amine product formation. Samples were taken in triplicate from three parallel reactions.
c] The enantiomeric excess was determined via chiral GC analysis using a Hydrodex-ß-TBDAc column (Macherey & Nagel).
2
[
So far, there has been no study, in which the influence of IPA on
transaminase activity has been explored to cover both aspects,
the acceptance of IPA as amine donor (requiring identification of
optimal pH values, concentration dependency as well as
mutations in the active site region) and the influence of IPA to
the overall protein stability as this amine donor also has solvent
effects on the enzyme. In this work, crucial amino acid residues
around the active site were identified for IPA acceptance and
also a systematic investigation was performed for a range of
ATA, different ketone substrates and different reaction
conditions with the aim of providing a more generic solution for
ATA-catalyzed asymmetric synthesis using IPA.
replaced by a glycine, which means that no movement of an
amino acid residue is required for substrate recognition. Ban-TA
showed provable activity towards propylamines (e.g. IPA) as
[
48]
amine donors. However, when the choice of substrates make
the coordination of acidic moieties obsolete, the role of the
‘flipping arginine’ (position 420 in 3FCR) is questionable and in
addition a strong positive charge at the entrance of the active
site tunnel was considered to be detrimental for the accessibility
of IPA on the PLP molecule. We used 3FCR-QM as template
and introduced at position 420 several amino acids with different
characteristics (hydrophobic, basic and acidic). The variants
were interrogated in our chosen asymmetric synthesis model
reactions (Scheme 1), because the GO-assay demands
coordination of glyoxylate and therefore was not suitable for the
purpose of a screening. The best-performing variant was the
mutation R420W (Table S2, SI), which we later introduced into
the 3FCR-QM-W59L variant. Additionally, we produced the
variant 3FCR-QM-W59L-R420A in order to provide an amino
acid residue at this position, which causes more space and low
interference at the entrance of the active site tunnel. Both
variants were compared in the mentioned model reactions with
IPA revealing a drastic improvement in conversion of 1a and 2a
compared to 3FCR-QM and 3FCR-QM-W59L (Table 1),
especially in case of 3FCR-QM-W59L-R420W. Remarkably, the
Recently, we reported the engineering of the ATA from
Ruegeria sp. TM1040 (PDB-ID: 3FCR) for the acceptance of
bulky ketones where the corresponding amines have
[
6,13,18]
pharmaceutical relevance.
The best variants from this
approach (e.g. one quadruple mutant 3FCR-Y59W-Y87F-
Y152F-T231A, 3FCR-QM) did not accept IPA well as the amine
[
13,18]
donor.
We already identified position 59 in 3FCR (according
to Protein Data Bank numbering) as very crucial for the activity
[
6,46]
towards aromatic and bulky substrates.
Interestingly, the
homolog position in the ω-transaminase from Ochrobactrum
anthropoi (OATA, W58) was reported to play a comparable role
in which the mutation OATA-W58L led to a better acceptance of
mutation at position 59 in 3FCR-QM
– which showed a
[
12]
aromatic ketones and amines. However, at the same time this
mutation was obviously responsible for a higher affinity towards
significant improvement in the GO-assay with IPA – did not lead
to a better conversion of 1a to 1b but at the same time a
detectable product formation of 2b. These results (especially for
IPA, which was proven by lower K
M
values and explained by
steric interference of W58 with one of the methyl groups of IPA.
Because of the notable sequence identity between OATA (PDB-
ID 5GHG) and 3FCR of approx. 43% we decided to focus again
on position 59 in the 3FCR scaffold. Starting from the variant
3
FCR-QM-W59L-R420W) are more meaningful when the
activities in kinetic resolution mode using rac-1b and rac-2b are
considered.
3
FCR-QM as template we saturated position 59 and screened
the NNK library against IPA using the glycine oxidase (GO)
[
47]
assay, as described previously.
It turned out that the only
variant with significant higher activity towards IPA as amine
donor contained the mutation 3FCR-QM-W59L which led to a
4
.6-fold higher activity compared to the template (see Table S1,
Supporting Information). Moreover, transaminases are
remarkable for their dual substrate recognition facilitated by a
flexible arginine residue, which is forming a salt bridge to the
carboxylate function of the respective substrate. It is highly
conserved in e.g. amino acid transaminases, ornithine
Scheme 2. Reactions studied for the investigation of conditions using IPA as
amine donor. Various donor-acceptor ratio and pH values were compared.
[
1,46]
transaminases and amine transaminases.
Notably, the ω-
amino acid transaminase from Bacillus anthracis (Ban-TA)
represents an exception as the ‘flipping arginine’ is naturally
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ChemCatChem
10.1002/cctc.201800936
COMMUNICATION
All three variants of 3FCR-QM showed lower activities for both
racemic amines (Table 1). For comparison, pentanal was used
as amine acceptor for all four enzymes because pyruvate was
not a suitable amine acceptor anymore mainly due to the lack of
were utilized in engineering and asymmetric synthesis
[
4–6,21,26,46,49–53]
approaches before.
For instance, Afu-TA (ATA
from Aspergillus fumigatus) and Spo-TA (ATA from Silicibacter
pomeroyi) were recently used for the production of halogenated
[
49]
[54]
the ‘flipping arginine’ (details in Fig. S1, SI). These results are
demonstrating that the introduced mutations in 3FCR-QM were
not responsible for a better substrate acceptance of 1a and 2a –
since mutations at position 59 have a great impact on the
reactivity towards bulky substrates in 3FCR – but rather they did
lead to a higher catalytic activity in asymmetric synthesis mode
with IPA as the amine donor. The best two variants 3FCR-QM-
W59L-R420A/W were subjected to the preparative scale
production of amines 1b and 2b in a 80 mg scale applying 0.5 M
IPA (see experimental section). The identity of the products was
chiral amines and Arth-TA (ATA from Arthrobacter sp.) for the
[
20,21]
synthesis of e.g. (R)-3,4-dimethoxy-amphetamine.
3FCR
wild-type and 3FCR-QM were excluded from these experiments
due to their low reactivity in pre-tests. We selected commonly
used aromatic benchmark substrates for the evaluation of amine
[
26,31,32,38]
donors
(3a and 4a, Scheme 2) and tested different pH
values as well as donor-acceptor ratio (Fig. 1). The amination of
acetophenone 3a to 1-phenylethylamine 3b is challenging due to
[
14,55]
the unfavored equilibrium,
so significant effects were
expected after reaction engineering. Additionally, we
1
13
confirmed via GC-MS, H- and C-NMR spectroscopy (see SI).
investigated one halogenated derivative of acetophenone (4a),
[
26]
since Cassimjee et al.
show-cased significant differences in
Next, we investigated the influence of reaction conditions on
the transaminase reactivity with IPA as amine donor. For that we
included our presented variants of 3FCR-QM and additionally
several commonly known wild-type ATA, which in pre-tests
showed significant conversion with IPA. Those wild-type ATA
conversion with substituted acetophenones and IPA using the
ATA from Chromobacterium violaceum (Cvi-TA). The donor-
acceptor ratio varied in a range of 5-fold – 100-fold excess of
IPA at the respective pH optimum of the ATA (Fig. S2, SI).
A
100
B
100
0
.025 M IPA
.05 M IPA
.1 M IPA
.25 M IPA
.5 M IPA
0.025 M IPA
0
0.05 M IPA
0.1 M IPA
0.25 M IPA
0
0
8
6
4
2
0
0
0
0
0
80
0
0.5 M IPA
60
40
20
0
A
A
A
A
-TA
-TA
th
TA
-TA
o
u-T
-T
th
-T
-T
59L
-W
i-
Cv
59L
W
i
o
u
420A
R
420A
R420W
Af
-
420W
R
Af
Ar
Cv
Sp
3
R
Ar
Sp
M
R-QM
-
Q
59L-
59L-
59L-
59L-
R
QM-W
QM-W
C
-W
C
QM-W
-
R
F
M
3F
-
-Q
CR-
R
R
C
C
C
3
F
3F
3F
3F
C
100
D
100
80
60
40
20
0
pH 7.5
pH 8
pH 7.5
pH 8
pH 8.5
pH 9
pH 8.5
pH 9
8
6
4
2
0
0
0
0
0
pH 10
pH 10
A
A
A
A
TA
TA
th-
TA
-TA
u-T
-T
th
-T
-T
59L
Afu-
i-
Cv
59L
i
o
o
QM-W
420A
R
420W
R
Af
Ar
Cv
Sp
3
-W
R
420A
R420W
Ar
Sp
M
-
Q
59L-
59L-
CR-
59L-
59L-
R
QM-
W
-W
-
W
M
C
-W
3F
M
-Q
F
M
-
Q
R
-
-Q
R
R
R
C
C
C
C
3
F
3F
3F
3F
Fig. 1. Asymmetric synthesis of 3b (A, C) and 4b (B, D) using IPA as amine donor. The influence of different donor-acceptor-ratio (A, B) and pH values
C, D) was investigated. Wild type ATA from Aspergillus fumigatus (Afu-TA), Arthrobacter sp. (Arth-TA), Chromobacterium violaceum (Cvi-TA),
(
Silicibacter pomeroyi (Spo-TA) and the presented variants of 3FCR (ATA from Ruegeria sp. TM1040) were used. A, B IPA was applied in the following
excess related to the amine acceptor: from left to right 5-fold, 10-fold, 25-fold, 50-fold and 100-fold (in a range of 0.025 – 0.5 M). The concentration of the
[
56]
ketone substrates was fixed at 5 mM. C, D Different pH values were set using Davies buffer : from left to right pH 7.5, pH 8, pH 8.5, pH 9 and pH 10.
-
1
The shown conversion levels are mean values out of duplicates. For single values see Supporting Information. General reaction conditions: 1 mg mL
purified enzyme, 5 mM 3a (substrate to enzyme ratio, s/e ~ 0.6 w/w) or 4a (s/e ~0.9 w/w), 5% (v/v) DMSO, 0.1 mM PLP, 50 mM HEPES/CHES buffer pH
7
2
.5 or 9 (according to each pH optimum, see Supporting Information), 30 °C. Samples were taken after 20 h and analyzed via gas chromatography with
-iodoacetophenone as internal standard.
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ChemCatChem
10.1002/cctc.201800936
COMMUNICATION
activity of the ATA were quantified via the initial activity assay
[
57]
(
acetophenone assay, Fig. 2). Interestingly, the majority of the
FCR variants were apparently affected by IPA incubation and
3
not any wild-type enzyme. But a considerable detrimental effect
due to IPA incubation was not obvious for any investigated ATA.
Additionally, protein melting points were determined in the
presence of IPA (see Table S5, SI) giving a similar result. This
experiment on ATA stability demonstrated that
a higher
tolerance towards IPA was not the reason for the better
conversion in the investigated reactions by 3FCR-QM variants.
Conclusively, the results from the pH and donor-acceptor-
ratio experiments revealed that both aspects should be
considered as enzyme as well as substrate dependent (or a
combination of both). Therefore no general guideline for an
optimal reaction setup can be derived. It appears that every ATA
reaction using IPA needs to be optimized relating to the
mentioned aspects since they likely have overlapping effects,
e.g. pH optima of the enzyme, pH/solvent stability and
protonation state of IPA. Through this reaction engineering even
wild-type enzymes could reach moderate to good activities with
Fig. 2. Volumetric activities (U/mL) determined via the initial activity assay
after incubation with IPA over 8 h (two concentrations were used as indicated).
Relative activities were normalized to an IPA free control experiment. Reaction
-
1
conditions: 0.05
– 0.1 mg mL purified protein, 1.25 mM (S)-PEA (1-
phenylethylamine), pyruvate or pentanal in equimolar ratio, 0.5 – 5% (v/v)
DMSO in CHES pH 9 (50 mM). The formation of acetophenone was quantified
at 245 nm at 30 °C. All measurements were performed in triplicate. The
compatibility of this assay under these conditions was ensured by previous
solvent exchange (Fig. S5 and S6, Supporting Information).
[
54]
IPA as shown here and also in previous works.
Thus, we
demonstrated a remarkable increase in catalytic activity towards
IPA as amine donor mutating amino acid residues around the
active site in the 3FCR-QM scaffold, namely positions 59 and
4
20. It has to be highlighted that the presented 3FCR-QM
variants exhibit a good to excellent performance in all shown
reactions, e.g. over a broad range of pH values and donor-
acceptor ratio. The mutation 59L is an important key mutation for
the catalytic activity towards IPA since the conversion in
reactions with 2a – 4a was already substantially increased. It
should be noted however that in terms of better IPA acceptance
the role of both positions (59 and 420) is definitely substrate
dependent. With the variant 3FCR-QM-W59L-R420W a general
approach for an improved IPA acceptance for the investigated
reactions was identified.
In case of acetophenone 3a the gradual increase of the IPA
concentration resulted in an increased conversion of 3a. The
only exception out of all investigated ATA was Cvi-TA showing a
decreased conversion level of 3a at high IPA concentrations. For
2
’-bromoacetophenone 4a no general statement in terms of IPA
excess effect could be made. While the results for Arth-TA
showed an increased activity at higher IPA concentrations, Spo-
TA and Cvi-TA were apparently less active the more IPA was
applied. But for the rest of the investigated ATA there was no
significant difference in conversion over the whole range of
applied IPA concentrations indicating a more favored equilibrium
situation. This was in line with previous results from literature
[
38]
mentioned above. All three 3FCR-QM variants showed indeed Experimental Section
the highest conversion of 4a, in case of 3FCR-QM-R420A and
3
FCR-QM-R420W even with quantitative conversion. The effect
All chemicals and kits were purchased either from Sigma Aldrich
Darmstadt, Germany), Roth (Karlsruhe, Germany), or Acros/Thermo
Fisher Scientific (Waltham, USA) in analytical grade. The ketones 1a and
a as well as the corresponding racemic amines (Scheme 1) were kindly
(
of the pH on the conversion level of 3a and 4a was investigated
over a range of pH 7.5 – 10, which was considered as the
common pH range for the most ATA reactions in literature. For
these experiments the IPA concentration was fixed to 0.5 M to
ensure no limitations here. Beside two significant exceptions
2
provided by F. Hoffmann-La Roche.
Enzyme expression, cell lysis and protein purification
(
Afu-TA and Arth-TA) the pH value did not influence the catalytic
activity of the ATA in both of the showed model reactions with
IPA. The two mentioned exceptions showed the trend that a
more basic pH is more beneficial for transamination reactions
with IPA since the concentration of unprotonated IPA is certainly
higher (Fig. 1C, 1D). Especially Arth-TA is interesting in this
manner since the best conversion of both 3a and 4a was
reached at pH 10 and in contrast the pH optimum of this enzyme
is at pH 8 (see SI). But the majority of the ATA showed a similar
activity in both reactions regardless of pH optima (see SI for all
ATA) and protonation state of IPA. To further support the results
from Fig. 1 we looked at enzyme stability in presence of IPA.
Beside the three presented variants of 3FCR-QM we chose
exemplary Cvi-TA and Spo-TA to cover all variations of catalytic
activity from Fig. 1. 3FCR wild-type and 3FCR-QM were
included for comparison. After incubation with IPA (final
concentration of 0.05 and 0.5 M) over 8 h at 30 °C the residual
Information about the plasmids containing genes of ATAs from
Chromobacterium violaceum, Silicibacter pomeroyi, Arthobacter sp.,
Aspergillus fumigatus and Ruegeria sp. TM1040 are given in Table S3
(Supporting Information). The protein expression was done in Terrific
-
1
-1
Broth (TB) media with 100 µg mL ampicillin or 50 µg mL kanamycin at
1
0
60 rpm and 20°C. After the optical density at 600 nm (OD600) reached
.5 – 0.7, expression was induced by adding 0.2 mM isopropyl β-D-1-
thiogalactopyranoside (final concentration). After 18 h the cultures were
centrifuged (4,000 x g, 15 min, 4 °C) and washed with lysis buffer
(
HEPES (50 mM pH 7.5), 0.1 mM PLP, 300 mM NaCl). Cell disruption
was performed via sonication using the Bandelin Sonoplus HD 2070
(8 min, 50% pulsed cycle, 50% power) on ice followed by centrifugation
in order to remove cell debris (12,000 x g, 45 min,
centrifuge). The supernatant containing the crude ATA was stored at
°C until use. Metal affinity chromatography was used to purify all
4 °C, Sorvall
4
enzymes with an Äkta purifier and a 5-mL HiTrap Fast Flow column (GE
Healthcare, Freiburg, Germany). Elution was mediated by the lysis buffer
additionally containing 300 mM imidazole. The desalting step was
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201800936
COMMUNICATION
performed using three HiTrap desalting columns in line (each 5 mL; GE
Healthcare, Freiburg, Germany) and lysis buffer without NaCl. The
protein concentration was determined via the Pierce BCA Protein Assay
Kit according to the manual.
for 5 min. For 4: initial temperature 100 °C, kept for 7.5 min, linear
-
1
gradient to 220 °C with a slope of 5 °C min , kept for 5 min. For retention
times see Supporting Information. The conversion of 1a and 2a was
determined by quantification of amine product formation via calculation of
the response factor. In case of 3a and 4a the same principle was
followed regarding substrate consumption. Each sample included the
mentioned internal standard and was set in relation to ATA-free control
experiments.
Point mutations via QuikChange mutagenesis
Variants of 3FCR-QM were produced using a modified version of the
QuikChange PCR method. Primers were designed with the desired
mismatches to provide the desired mutations. For each PCR, Pfu buffer,
Preparative scale synthesis of 1b and 2b
−
1
0
0
.2 mM dNTPs, 0.2 ng µL parental plasmid, 0.2 µM of each primer and
.2 µL of Pfu Plus! DNA polymerase were applied. DMSO
A
The conversion of 1a and 2a to the corresponding amine was performed
in preparative scale. 87 mg 1a (using 3FCR_QM_W59L_R420A) and
84 mg 2a (using 3FCR_QM_W59L_R420W) were applied respectively in
an Erlenmeyer flask and dissolved in DMSO (5% final concentration).
CHES buffer (50 mM final, pH 9) and isopropylamine (0.5 M final
concentration) were added under stirring. The pH was adjusted with
aqueous HCl. In the end the reaction was started by addition of
concentration of 3% (v/v) was set. The amplification was performed as
follows: (a) 94 °C, 2 min; (b) 25 cycles: 94 °C, 30 s; 55 °C or 60 °C, 30 s;
7
2 °C, 7:05 min (c) 72 °C, 14 min. The PCR product was digested with
−
1
DpnI (20 µL mL ) for 2 h at 37 °C and the restriction enzyme was
inactivated by incubation at 80 °C for 20 min. Chemo-competent Top10
E. coli cells were transformed with the PCR product. After confirmation of
the correct sequence the plasmids were isolated from Top10 and chemo-
competent E. coli BL21 (DE3) cells were transformed for protein
expression as described above.
-
1
1 mg mL enzyme which led to a final working volume of 0.2 L and a
final substrate concentration of 2 mM (1a s/e ~ 0.43, 2a s/e ~ 0.42). The
reaction mixture was incubated for 48 h at 30 °C under agitation. For the
quantification of the conversion samples were taken, extracted as
described above and analyzed via GCMS. The reaction was stopped
when no further conversion was observed during reaction monitoring (for
Determination of transaminase activity
1
a 53%, for 2a 27%). The following reaction workup was done: After
The characterization of the ATA was done via the initial activity assay
[
57]
reaction quenching with 10 mL 3 M NaOH to a pH of >12, an extraction
with 1x 0.2 L and 1x 0.1 L hexane was performed in a separation funnel.
(
acetophenone assay) according to Schätzle et al.
with slight
modifications. In the reaction solution the concentrations of the amine
donor ((R)-/(S)-1-PEA 3b, rac-1b or rac-2b) and the acceptor pyruvate or
pentanal was set to 1.25 mM or as indicated in 0.5% - 10% (v/v) DMSO.
Briefly, 10 µL of a pre-diluted ATA solution was mixed with the respective
buffer (according to the pH optimum of each ATA, HEPES or CHES
buffer) and the reaction was initiated by the addition of the 4-fold
concentrated stock of reaction solution. The formation of the
corresponding ketone was quantified at 245 nm (2a and 3a) or 265 nm
4
The combined organic layers were dried over anhydrous MgSO and
evaporated under vacuum to a volume of 0.5 mL. The crude reaction
product was applied to a silica column with ethyl acetate as mobile phase.
The fractionation monitoring was done via TLC. Fractions containing the
respective amine product were pooled and evaporated under vacuum
until dryness. The consistency of the amine products was a yellow oil.
Each product was confirmed via GCMS and 10-12 mg each were
1
13
subjected to H- and
C-NMR spectroscopy (see Supporting
(
1a), respectively using the Tecan Infinite M200 Pro (Crailsheim,
Information). 1b, 53% conv., 35% isolated yield (not optimized), 55.5%ee.
2b, 27% conv., 33% isolated yield (not optimized), 98%ee.
Germany) at 30 °C. One unit (U) of ATA activity was defined as the
-
1
-1
formation of 1 µmol of 1a – 3a per minute (1a ε= 16.56 M cm , 2a ε=
-
1
-1
-1
-1
9
.65 M cm , 3a ε= 12 M cm ). All measurements were performed in
ATA stability in the presence of IPA
triplicate. The pH optimum of each ATA was determined by using
[
56]
Davies buffer with a pH value as indicated.
Enzyme stability was investigated by incubation samples of ATA with IPA
(
0, 0.05 and 0.5 M final concentration) for 8 h at 30 °C at 950 rpm
shaking. Afterwards a solvent change was performed via PD columns®
GE Healthcare, Freiburg, Germany) according to the manual. Fractions
Asymmetric synthesis of the chiral amines 1b – 4b
(
Biotransformations were performed in 0.25 mL scale using 1.5 mL glass
vials at 30 °C and 950 rpm shaking. The reaction mixtures contained
of 0.5 mL were taken and the most active one was subjected to further
investigations. The residual enzyme activity was measured via the initial
activity assay. For reaction conditions see figure capture. Additionally,
-
1
1
mg mL purified ATA, 5 mM ketone, 30% DMSO as co-solvent, the
respective concentration of IPA (from a 4 M IPA-HCL stock solution, pH
) in HEPES, CHES (50 mM) or Davies buffer as indicated. The final pH
melting points T
M
of each subjected ATA in presence of 0, 0.05 and
7
0
.5 M IPA (final concentration) were determined using the Prometheus
was checked. Additionally, control experiments with desalting buffer
instead of enzyme were performed. After 20 h incubation, the reaction
was quenched by adding 3 M NaOH (resulting in pH ≥12). Samples for
gas chromatography (GC) analysis were taken immediately after
quenching.
®
NT.48 device from nanotemper (see Supporting Information). The
-
1
protein concentration was set to 1 mg mL in HEPES buffer pH 7.5 (50
-
1
mM) including 50 µM of PLP. The heating rate was set to 0.5 °C min
from 20 – 95 °C. Inflection points (or melting points, respectively) were
determined by the first derivative of the measured fluorescence at a
3
30/350 nm ratio.
GC analysis
Samples of 100 µL were withdrawn for chiral GC analysis and extracted
with 300 µL of ethyl acetate containing 1 mM 2’-iodoacetophenone as
internal standard for quantification. The organic layers were dried over
Acknowledgements
anhydrous MgSO
4
and derivatized (when necessary) with N-Methyl-bis-
We thank the Bundesministerium für Bildung und Forschung
trifluoroacetamide (MBTFA) by adding 7.5 µL of the commercial stock
solution to 100 µL of the organic layer and incubation at 60 °C for 30 min.
Afterwards, the samples were analyzed immediately using the Hydrodex-
ß-TBDAc column (Macherey & Nagel). For the analysis of substances 1
and 2 the following temperature gradient program was established: initial
temperature 140 °C, kept for 15 min, linear gradient to 180 °C with a
(
Grant 01DN14016) for financial support. Prof. Dr. de Souza
thanks the Alexander-von-Humboldt foundation for a Capes-
Humboldt research fellowship.
Keywords: Amine transaminases, biocatalysis, chiral amines,
-
1
slope of 15 °C min , kept for 35 min, linear gradient to 220 °C with a
enzyme engineering, isopropylamine
-
1
slope of 15 °C min , kept for 10 min. For 3: initial temperature 120 °C,
-
1
kept for 5 min, linear gradient to 220 °C with a slope of 10 °C min , kept
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ChemCatChem
10.1002/cctc.201800936
COMMUNICATION
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ChemCatChem
10.1002/cctc.201800936
COMMUNICATION
Improved variants for higher
catalytic activity towards
isopropylamine (IPA) as
amine donor were created by
Ayad W.H. Dawood, Martin
S. Weiß, Christian Schulz,
Ioannis V. Pavlidis, Hans
Iding, Rodrigo O.M.A. de
Souza, Uwe T. Bornscheuer*
protein
engineering
and
applied in the asymmetric
synthesis of industrial rele-
vant amines. A systematic
investigation of pH and an
excess of IPA indicates the
necessity of substrate- and
ATA-dependent
Page No. – Page No.
Isopropylamine as Amine
Donor in Transaminase-
Catalyzed Reactions:
Better Acceptance through
Reaction and Enzyme
Engineering
optimizations.
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