Michael J. Abrahamson et al.
FULL PAPERS
À1
À1 [23]
NADH (e340 =6220M cm ).
For reductive amination, Enantioselectivity and Conversion
reactions were performed in 225 mM NH Cl/NH OH buffer
4
4
A reaction was carried out in 100 mL of 225 mM NH Cl/
4
at pH 9.6, with 200 mM NADH and 20 mM of the ketone
substrate, unless otherwise specified. For oxidative deamina-
tion, reactions were performed in 0.1M NaHCO /Na CO
3
buffer at pH 10.0, with 1 mM NAD with 10 mM of the
amine substrate of interest. All reactions were performed at
NH OH buffer (pH 9.6) containing 20 mM PFPA, 22 mM
4
+
glucose, 2 mM NAD , 17.5 mg PheDH-AmDH and 200 U
3
2
+
of glucose dehydrogenase 103 at 258C. The conversion was
allowed to continue for 48 h, after which the reaction mix-
ture was adjusted to pH 13 with 5 mL of 10N NaOH and ex-
tracted with methyl tert-butyl ether. The organic extract was
2
58C unless otherwise specified.
dried with anhydrous MgSO and concentrated under re-
4
Mutagenesis
duced pressure to give of FPPAm; yield: 212 mg (93.8%
conversion, 73.9% yield).
After the first 10 rounds of mutagenesis, three mutations
were previously identified to be most influential in creating
reductive amination activity in a LeuDH-based scaffold;
K67M, E113V, and N261V. Analogous mutations to these
were identified through sequence alignment and applied to
a similar amino acid dehydrogenase scaffold, phenylalanine
dehydrogenase (PheDH) from Bacillus badius (48% identi-
ty, 66% similarity). Each of the point mutations, (K77M,
T123V, N276V) as well as combinations of these mutations,
was evaluated for amination activity.
Mutant libraries were generated using overlap extension
PCR. After identification of mutational sites, primers were
designed according to the guidelines of the QuikChange
Site Directed Mutagenesis Protocol. These primers were
then used in the overlap extension protocol described in
Molecular Cloning: A Laboratory Manual.
1
The H NMR spectrum of the isolated product was consis-
tent with the structure of (R)-(À)-1-(4-fluorophenyl)-propyl-
[1]
[25]
1
2-amine and agreed with reported data.
H NMR
(400 MHz, CDCl ): d=7.26–6.95 (m, 4H), 3.12 (m, 1H),
3
2.67 (dd, 1H, 6.3 Hz, 13.2 Hz), 2.49 (dd, 1H, 6.3 Hz,
13.2 Hz), 1.10 (d, 3H, 6.4 Hz).
Confirmation of the (R)-selectivity was achieved through
polarimetry, since single enantiomers of (4-fluorophenyl)-
propyl-2-amine were not available. Optical rotation was
measured on a Rudolph Autopol III polarimeter at 589 nm
2
D
5
resulting in a specific rotation of [a]
MeOH).
: À38.4 (c 0.46,
ꢄ
[25]
[24]
[22]
After muta-
tion, the resulting mutated gene was digested using NdeI
and XhoI, and ligated into pET17b. The resulting plasmids
were transformed into BL21 (DE3) E. coli competent cells.
Acknowledgements
M.J.A. gratefully acknowledges support from a GAANN fel-
lowship by the US Dept. of Education and from NSF I/
UCRC grant 0969003 to the Center for Pharmaceutical De-
velopment. Support for the Genetix Colony picker by the
NSF-MRI program 0320786 is gratefully acknowledged.
Library Screening
After mutagenesis, colonies were robotically picked using
a Genetix QPix2 colony picker and expressed in microtiter
plates containing 250 mL of MagicMediaꢃ media for 18 h at
378C. After expression, the plates were pelleted at 1000 rpm
for 30 min and frozen until screening at À808C. As the ex-
pression plates thawed, the cell pellets were gently vortexed
with 30 mL of B-PER to uniformly resuspend and lyse the
cells. The crude cell lysate was split in 10 mL aliquots into
two plates; a reaction and background plate. A reaction
mixture (200 mL) containing 10 mM PFPA and 200 mM
References
[
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24, 4036; Angew. Chem. Int. Ed. 2012, 51, 3969.
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NADH in 225 mM NH Cl/NH OH buffer was added to
4
4
each well. The same mixture lacking PFPA was added to the
background plate. Absorbance measurements at 340 nm
began immediately and continued periodically over the
course of 1.5 h.
Active variants were identified by the rate of change in
absorbance at 340 nm, corresponding to the fastest activity.
The wells exhibiting the fastest rate change over that of the
background plate were selected for further characterization.
Some variants consumed the cofactor too quickly (<5 min)
to be observed over multiple time points, and gave low ab-
sorbance at 340 nm in the initial time point. These variants
were included for further characterization. The successful
variants were sequenced, and each unique sequence was ex-
pressed in pET28a/BL21 for his-tag purification and deter-
mination of kcat and KM values with PFPA.
1
0; f) G. W. Huisman, J. Liang, A. Krebber, Curr. Opin.
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ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 0000, 000, 0 – 0
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