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K. Fesko et al. / Journal of Molecular Catalysis B: Enzymatic 96 (2013) 103–110
was initiated by addition of 0.5 mM IPTG (isopropyl -d-1-
thiogalactopyranoside) to OD600 ∼0.8 cultures, and cultivation
was continued at 25 ◦C for 20 h. Protein expression and localization
in the supernatant was confirmed by SDS-PAGE. The cells were har-
vested, resuspended in cold buffer (50 mM potassium phosphate
buffer pH 7.5 containing 0.1 M PLP) and disrupted by sonication
(Branson Sonifier S-250; 6 min, 80% duty cycle, 7 output control).
The cell lysate was centrifuged for an hour at 50,000 g to remove
unbroken cells and insoluble material. The cell free lysate was fil-
tered through a 0.45 m syringe filter and concentrated to about
40 mg/ml using Vivaspin 20 Centrifugal Filter Units (10,000 Da
molecular-weight cut-off; Sartorius). The total protein concentra-
tion was analyzed by Bradford protein assay (Biorad).
Scheme 1. Asymmetric synthesis of (S)- and (R)-amines using -transaminases.
the production of an amine. Among the most often used methods
to accomplish this endeavour is the removal of formed pyruvate
from the reaction mixture using either lactate dehydrogenase [25]
or alanine dehydrogenase [9]. Pyruvate decarboxylase [26] and ace-
tolactate synthase [12] were also employed for the degradation of
pyruvate to shift the equilibrium towards the amination reaction.
On the other hand, the use of several enzymes in one pot is not
led to a more favourable product equilibrium compared to ala-
nine. Inexpensive 2-propylamine can be used as alternative donor
[19,27–29] and the formed by-product acetone might be removed
principle, chiral amines such as 1-aminoindane, 1-aminotetralin,
1-phenylethylamine (1-PEA) or achiral 1-phenylmethanamine are
thermodynamically favourable for the transamination and good
donors for -TA, however due to high costs they cannot be applied
on a large scale [30]. Thus, the search for alternative amino donors,
2.3. General procedure for -transaminase catalyzed reactions
Sodium phosphate reaction buffer (50 mM, pH 7.5) containing
0.1 mM PLP was mixed with the ketone substrate (10 or 50 mM)
and the appropriate amino donor (0.1–0.5 M). The pH was adjusted
to 7.5 with phosphoric acid before adding the enzymes. The reac-
tions were started by adding 50 l of cell-free extract (CFE, contains
approximately 2 mg of protein) and then shaken at 30 ◦C for 24 h.
2.4. Analysis of reactions
2.4.1. Determination of conversions
50 l from the reaction mixture were added to 950 l of an ace-
tonitrile/water mixture (50:50) containing 0.01% formic acid. Then
the solution was centrifuged at 13000 rpm for 5 min and analyzed
by rp-HPLC. HPLC-analysis was carried out on a Purospher Star
RP C18 (250 × 4.0 mm, 5 m, Merck, Darmstadt) column at 30 ◦C
and a flow rate of 0.80 ml/min using varying mixtures of eluent A
(water + 0.01% HCOOH) and eluent B (acetonitrile). UV detection at
210 nm was applied. Where necessary, appropriate response fac-
tors obtained with reference compounds were used to calculate
the conversions.
Herein, we present our research on three wild-type -
transaminases (S)-TA Pseudomonas aeruginosa (Pae) [31], (S)-TA
Paracoccus denitrificans (Pde) [22,32,33] and (R)-TA Aspergillus ter-
reus (Ate) [5,34]. The enzymes were applied for the synthesis of
aromatic chiral amines using different amine donors. Moreover,
the substrate spectrum, enzyme inhibition and influence of the
reaction conditions on the conversion and enantioselectivity were
investigated. Proper selection of the amino donor/amino acceptor
pair is the essential factor in the successful transamination.
2.4.2. Determination of enantiomeric excesses
The enantiomeric excess of amines was determined after deriva-
tization with Marfey’s reagent as following: 20 l of the reaction
mixture were mixed with 50 l of an N-␣-[2,4-dinitrophenyl-5-
fluorophenyl]-l-alanine amide solution (1% (w/v) in acetone) and
10 l saturated NaHCO3. After incubation of the mixture at 40 ◦C for
1 h, 10 l 2 M HCl and 920 l acetonitrile were added and the sam-
ple was centrifuged for 5 min prior to analysis. HPLC-analysis was
carried out on a Purospher Star RP C18 (250 × 4.0 mm, 5 m, Merck,
Darmstadt) column at 30 ◦C and a flow rate of 1.00 ml/min using
a 50:50 mixture of eluent A (water + 0.01% HCOOH) and eluent B
(acetonitrile). UV detection was performed at 338 nm.
2. Experimental
2.1. General
All chemicals were purchased from Sigma Aldrich, Fluka, Acros
Organics, TCI Europe or Alfa Aesar, unless stated otherwise. All
enzymes for genetic work were purchased from Fermentas (now
part of Thermo Scientific Molecular Biology). HPLC analyses were
conducted on an Agilent 1100 or Agilent 1200 instrument. More
detailed information is provided in the supporting information.
2.4.3. Determination of enzyme inhibition by pyruvate
2.2. Production of -transaminases
The reactions were performed in
and the mixture consisted of 5–50 mM pyruvate, 50 mM 1-
phenylethylamine, 0.1 mM PLP and 0.03 mg purified (S)-TA Pde in
50 mM phosphate buffer pH 7.5. The initial reaction rates were
measured spectro-photometrically by monitoring the formation of
1-phenylethanone at 300 nm (ε = 0.28 cm2 mol−1) [35].
The gene (gene ID: 115385557) encoding the -(R)-
transaminase (XP 001209325) from A. terreus was ordered
codon-optimized for E. coli from GeneArt (Life Technologies). The
gene was recloned into the expression vector pMS470ꢀ8-vector
via the restriction sites NdeI and HindIII. The genes (gene ID:
9951072 and 119386691) encoding the -(S)-transaminases
(AE004893) from P. aeruginosa and (YP 917746) from P. denitri-
ficans were PCR amplified from plasmids provided by DSM and
ligated via the restriction sites NdeI and HindIII into pMS470ꢀ8-
vector. The three constructs were transformed into E. coli TOP10F’
cells (Invitrogen).
3. Results and discussion
3.1. Investigation of the donor range
The choice of a proper amino donor for the asymmetric synthe-
sis of chiral amines with -TAs is of great importance. The amino
donor must be efficient in the desired transformation, inexpen-
sive, well soluble and the corresponding carbonyl product should
E. coli TOP10F’ cells harbouring pMS470-Ate, pMS470-Pae or
pMS470-Pde were grown in LB medium supplemented with ampi-
cillin (80 g/ml) at 37 ◦C. Expression of recombinant protein