Tailoring D-amino acid oxidase from the pig kidney to r-stereoselective amine oxidase and its use in the deracemization of α-methylbenzylamine

Article abstract of DOI:10.1002/anie.201308812

The deracemization of racemic amines to yield enantioenriched amines using S-stereoselective amine oxidases (AOx) has recently been attracting attention. However, R-stereoselective AOx that are suitable for deracemization have not yet been identified. An R-stereoselective AOx was now evolved from porcine kidney D-amino acid oxidase (pkDAO) and subsequently use for the deracemization of racemic amines. The engineered pkDAO, which was obtained by directed evolution, displayed a markedly changed substrate specificity towards R?amines. The mutant enzyme exhibited a high preference towards the substrate α- methylbenzylamine and was used to synthesize the S?amine through deracemization. The findings of this study indicate that further investigations on the structure-activity relationship of AOx are warranted and also provide a new method for biotransformations in organic synthesis. A change in selectivity: An engineered porcine kidney D-amino acid oxidase (pkDAO) with markedly changed substrate selectivity towards R?amines was obtained by directed evolution. The mutant enzyme exhibited a high preference towards the substrate α-methylbenzylamine and was used to synthesize the S-configured amine through deracemization.

Full text of DOI:10.1002/anie.201308812

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Angewandte
Communications
DOI: 10.1002/anie.201308812
Enzymatic Deracemization
Tailoring d-Amino Acid Oxidase from the Pig Kidney to R-Stereose-
lective Amine Oxidase and its Use in the Deracemization of a-
Methylbenzylamine**
Kazuyuki Yasukawa, Shogo Nakano, and Yasuhisa Asano*
Abstract: The deracemization of racemic amines to yield
enantioenriched amines using S-stereoselective amine oxidases
(AOx) has recently been attracting attention. However, R-
stereoselective AOx that are suitable for deracemization have
not yet been identified. An R-stereoselective AOx was now
evolved from porcine kidney d-amino acid oxidase (pkDAO)
and subsequently use for the deracemization of racemic
amines. The engineered pkDAO, which was obtained by
directed evolution, displayed a markedly changed substrate
specificity towards R amines. The mutant enzyme exhibited
a high preference towards the substrate a-methylbenzylamine
and was used to synthesize the S amine through deracemiza-
tion. The findings of this study indicate that further inves-
tigations on the structure–activity relationship of AOx are
warranted and also provide a new method for biotransforma-
tions in organic synthesis.
position, such as a-methylbenzylamine (MBA).[5] Several
studies have examined the actions of AOx on chiral
S amines.[2] Turner et al. reported engineered S-stereoselec-
tive flavin-dependent monoamine oxidase (MAO) variants
from Aspergillus niger for the deracemization of racemic
amines to produce chiral primary, secondary, and tertiary
amines.[2a,b,e,f] The catalytic activity of these mutants towards
chiral primary amines was shown to be higher than that
towards simple primary amines. Leisch and co-workers
successfully synthesized an R amine by deracemization
using an S-stereoselective cyclohexylamine oxidase from
Brevibacterium oxydans IH-35A.[2c,d] More recently, Kohler
et al. obtained secondary cyclic R amines from the corre-
sponding racemic amines or imine precursors through a cas-
cade reaction with a metalloenzyme under mild conditions.[6a]
The reaction consisted of two steps, namely amine oxidation
by mutant MAO and reduction of the imine with an artificial
transfer hydrogenase[6b] instead of a harsh chemical reductant.
However, R-stereoselective AOx that are suitable for dera-
cemization have not yet been identified. TPQ-dependent
AOx from Escherichia coli and Klebsiella oxytoca were shown
to preferentially oxidize the R enantiomer of amphetamine
with moderate enantiomeric ratios (corresponding to an
E value of ca. 15),[7] but they are not suitable for deracemiza-
tion reactions because the cofactor TPQ and the intermediate
imine formed a covalent bond. The purpose of this present
study was to evolve a flavin-dependent porcine kidney d-
amino acid oxidase (pkDAO) into an R-stereoselective AOx
and apply it to the deracemization of racemic amines.
AOx with R stereoselectivity have not been reported
previously, but S-stereoselective flavin-dependent AOx
belonging to the AOx family proteins, including l-amino
acid oxidase (LAO), polyamine oxidase, and spermine
oxidase, have been described. We targeted the DAO family
of proteins and pkDAO in particular as a starting enzyme for
directed evolution, because when studying their primary
structures, we observed that the DAO family of proteins,
including DAO, glycine oxidase, and sarcosine oxidase, may
have diverged from a common ancestral protein; therefore,
the potential protein structure of R-stereoselective AOx
should resemble that of DAO. We speculated that it may be
possible to tailor-make AOx from typical DAO enzymes, such
as pkDAO.
T
he efficient enzymatic synthesis of chiral amines, which are
important building blocks for pharmaceuticals and agro-
chemicals, has been the focus of academia and industry.
Lipase, an R- or S-stereoselective transaminase, has mainly
been used to examine the enzymatic synthesis of chiral
amines.[1] The deracemization of a racemic amine to obtain
the R-configured amine has been reported using an S-
stereoselective amine oxidase and a chemical reductant.[2]
Amine oxidases (AOx) catalyze the oxidative deamination
of amines to form the corresponding aldehyde, hydrogen
peroxide, and ammonia via an imine intermediate. AOx can
be classified into two groups based on the type of cofactor in
the active site, namely copper-containing topaquinone
(TPQ)-dependent AOx[3] and flavin-dependent AOx.[4]
These AOx preferentially oxidize simple straight-chain pri-
mary amines, such as butylamine, phenylethylamine, and
dopamine, rather than amines with a chiral center at the a-
[*] K. Yasukawa, Dr. S. Nakano, Prof. Dr. Y. Asano
Biotechnology Research Center and Department of Biotechnology
Toyama Prefectural University
5180 Kurokawa, Imizu, Toyama 939-0398 (Japan)
and
Asano Active Enzyme Molecule Project, ERATO, JST
5180 Kurokawa, Imizu, Toyama 939-0398 (Japan)
E-mail: asano@pu-toyama.ac.jp
[**] This work was supported by the Asano Active Enzyme Molecule
Project, ERATO, JST. X-ray data were collected at the synchrotron
facilities of the Photon Factory (PF) using beamline BL17A
(2013G004). We are grateful to the beamline staff and Dr. Yamada
for their assistance with the experiments.
pkDAO was the first identified mammalian flavoprotein
that catalyzes the oxidative deamination of a-amino acids
with strict R stereoselectivity to form the corresponding a-
keto acids, ammonia, and hydrogen peroxide, but it does not
act on simple amines. The structure of flavin-dependent
pkDAO complexed with benzoate as an inhibitor was
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201308812.
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previously revealed (PDB code: 1AA8).[8] The overall
structure of pkDAO was markedly different from that of
the AOx family of proteins. On the other hand, the substrate-
binding site of pkDAO and LAO may be considered as mirror
images of each other, and their catalytic mechanisms have
many similarities.[4] The carboxylate group of the benzoate
interacts with the guanidine moiety of Arg283, and the
hydroxy oxygen atom of Tyr228 acts as the carboxylate
binding site in cooperation with Arg283 in pkDAO. This
arginine residue is conserved in several d- or l-amino acid
oxidases from different sources. Therefore, the residues
Tyr228 and Arg283 in the catalytic site were chosen as the
target sites for mutation to improve substrate specificity. The
pkDAO gene was synthesized by assembly PCR and
expressed in E. coli bacteria. A first round of saturation
mutagenesis of the Tyr228 and Arg283 residues was per-
formed, and the resulting mutant libraries were screened in
the oxidation of (R/S)-a-MBA ((R/S)-1). A positive clone was
determined by a colorimetric assay that measured amine
oxidase activity. The positive mutant could not be obtained
from the saturation mutagenesis library of Tyr228. Twenty
variants with oxidation activity toward (R/S)-1 were obtained
by screening the saturation mutagenesis library of residue
Arg283. The residue Arg283 was found to have been
replaced by either Gly, Ala, or Cys in the positive mutant
enzymes that were obtained. The mutants R283G, R283A,
and R283C catalyzed the oxidation of the R enantiomer of
(R/S)-1. These mutants were used as parents for the second
round of saturation mutagenesis of Tyr228 and screening. The
resulting mutants, including Y228L/R283G, Y228L/R283A,
and Y228L/R283C, were obtained by screening for an
oxidative activity that is higher than that of the parent
enzymes (Table 1). These mutants also showed highly stereo-
selective oxidase activity towards the R enantiomer of (R/S)-
1 only. Finally, the mutant pkDAO Y228L/R283G was
selected for further investigations because it showed the
highest activity towards (R/S)-1.
configured amino acids, such as phenylalanine, proline,
methionine, and alanine. Its specific activity was four times
higher than that of purified wild-type pkDAO (4.9 Umgꢀ1,
with (R)-phenylalanine as the substrate). The reaction profile
of the kinetic resolution of (R/S)-1 (10 mm) to (S)-1 using the
purified enzyme is shown in Figure 1a. The initially present
Figure 1. Time course of the kinetic resolution (a) and the deracemiza-
tion (b) of (R/S)-1 using the mutant pkDAO Y228L/R283G. a) The
kinetic resolution of (R/S)-1 (10 mm) was carried out with mutant
pkDAO (1.5 U) in KPB (100 mm, pH 8.0) at 308C. b) Enzymatic
conversion of (R/S)-1 into (S)-1 by deracemization. The reaction
mixture (total volume 1.0 mL) was composed of KPB buffer (100 mm;
pH 8.0), (R/S)-1 (5 mm), NaBH4 (100 mm), and the mutant enzyme
(0.5 U) at 308C. The enzyme was added to initiate the reaction. The
*
*
amounts of (S)-1 and (R)-1 are denoted by and , respectively.
(R)-1 completely disappeared from the reaction mixture
within two hours, and the enantiopurity of the remaining (S)-
1 reached 99% ee. This result indicates that the mutant
enzyme is a novel R-stereoselective amine oxidase that could
be applied to the production of chiral S-configured amines by
kinetic resolution. Other mutants also showed completely R-
stereoselective activity toward (R/S)-1. The mutant enzyme
also exhibited a strong preference for MBA derivatives
(especially for 1, 8, and 15), whereas the enzyme hardly
oxidized chiral aliphatic primary amines (12 and 13), simple
primary amines (20 and 21), or chiral secondary R amines (13;
Figure 2). However, S amines (1–8, 14) were found to be
unsuitable substrates. The stereoselectivity of the mutant
pkDAO towards (R/S)-1, (R/S)-3, and (R/S)-4 was determined
by HPLC on a chiral stationary phase. Furthermore, both
enantiomers of several amino acids, such as 22, 23, 24, and
glycine, could not be used as substrates for this enzyme. A
comparison of the mutant enzyme with wild-type pkDAO
showed that they had the same properties (preferring pH 9
and 458C) except for their heat stability: The mutant enzyme
was stable at 558C, whereas the wild-type enzyme was stable
only up to 458C.[9]
This mutant pkDAO (Y228L/R283G) was purified and
characterized from recombinant E. coli bacteria (JM109).
The specific activity of the purified enzyme was 21.5 Umgꢀ1
for (R)-1 as the substrate, and the enzyme acted as a strictly R-
stereoselective amine oxidase in the presence of (R/S)-1. This
mutant lost its ability to catalyze the oxidation of R-
Table 1: Comparison of the activities of the pkDAO variants.[a]
Relative activity [%]
Variant pkDAO
(R)-Phe
(R)-MBA
Wild-type pkDAO has been used for the deracemization
of a-amino acids to form S-configured a-amino acids.[10] The
Y228/R283G mutant enzyme was also capable of stereo-
inversion of an R-configured amine using a chemical reduc-
tant such as NaBH4. The mutant enzyme lost its activity under
the harsh deracemization reaction conditions, which include
the use of NaBH4 at high temperature. The activity of the
enzyme was almost lost when it was incubated at 458C for
30 minutes in the presence of NaBH4 (100 mm). Milder and
more stable chemical reductants, such as NaCNBH3 and
H3N·BH3, were previously shown to be suitable for use in
deracemization reactions using AOx.[2,10c,d] However, the
wild-type
R283G
R283A
100
0
0
0
0
0
0
0
0
26
14
14
146
20
38
0
R283C
Y228L/R283G
Y228L/R283A
Y228L/R283C
Y228L
[a] The activity of (R)-phenylalanine (Phe) that corresponds to
0.11 Umgꢀ1 was taken as 100%. The reaction mixture (total volume:
1.0 mL) was composed of KPB (100 mm; pH 8.0), the substrate
(10 mm), and an appropriate amount of the cell-free extract.
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reaction is shown in Figure 1b. Conver-
sion of (R)-1 into (S)-1 was quantitative,
and (R/S)-1 had been completely con-
verted into (S)-1 (99% ee) with no
detectable side product formation after
three hours. Approximately 35% of the
S amine was lost during the purification
process, as it was isolated in 65% yield.
To the best of our knowledge, this is the
first study that examines the synthesis of
S amines by a deracemization process
using an R-stereoselective amine ox-
idase. This mutant enzyme is useful for
the synthesis of the S enantiomer of
amines, as it not only provides a method
for kinetic resolution, but also catalyzes
the deracemization process.
The exchange of a single amino acid
to obtain the mutant R283G led to
a marked change in the substrate spe-
cificity of the enzyme from amino acid
oxidase to amine oxidase. We deter-
mined the crystal structure of the
pkDAO mutant Y228L/R283G (PDB
code: 3WGT). The crystal structure of
the pkDAO mutant that binds (R)-1 was
determined at a resolution of 1.88 ꢀ
(Supporting Information, Table S5), and
the structure of the active site is shown
in Figure 3a. The electron density of
Figure 2. Substrate specificity of mutant pkDAO. Enzyme activities were measured as described
in the Experimental Section. The activity of (R)-1 that corresponds to 21.5 Umgꢀ1 was taken as
100%. The italicized numbers below the structures indicate the relative activity. [a] Substrate
specificity of wild-type pkDAO. [b] See Ref. [9].
conversion of (R)-1 (1 mm) into (S)-1 was lower with
NaCNBH3 (8.5% yield) and H3N·BH3 (48%) than with
NaBH4 (96%). The use of NaCNBH3 or H3N·BH3 led to the
formation of the undesired ketone, and an alcohol was then
obtained as the main product by reduction of the ketone. On
a preparative scale, the synthesis of (S)-1 from racemic 1 by
deracemization was performed at 308C under the following
optimized conditions: KPB buffer (100 mm; pH 8.0) contain-
ing (R/S)-1 (5 mm), NaBH4 (100 mm), and purified enzyme
(300 U). The typical time course of the deracemization
(R)-1 was confirmed to be located on the re side of flavin
adenine dinucleotide (FAD; Figure 3a). A hydrophobic
cavity, which has enough space to accommodate the phenyl
ring of (R)-1 (Figure S5a), was created near the xylene ring of
FAD in the Y228L/R283G mutant. In this location, the
benzene ring of the substrate was sandwiched between the
xylene ring of FAD and the para-hydroxyphenyl group of
Tyr224 and experienced p–p stacking interactions with the
xylene ring and the para-hydroxyphenyl group of Tyr224
(Figure S5b). This arrangement is different from that for the
Figure 3. a) The active site of the pkDAO mutant Y228L/R283G with bound (R)-MBA. The carbon atoms of (R)-1 (or (R)-MBA) are shown in
green. The carbon atoms of pkDAO and FAD are colored in gray. All hydrogen bonds were shorter than 3.4 ꢀ. The 2FoꢀFc Fourier maps (blue)
were contoured at 0.8 s. b) Proposed mechanism for (R)-1 bound to the pkDAO mutant Y228L/R283G. c) Proposed mechanism for (S)-1 bound
to the pkDAO mutant Y228L/R283G. The reaction mechanism for the mutant was proposed in analogy to the reaction mechanism that was
already suggested for wild-type pkDAO.[11]
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The mixture was then incubated at 308C for 3 h. The reaction mixture
binding of the inhibitor benzoate to wild-type pkDAO, which
was observed by crystal structure analysis; in this case, the
phenyl group of the benzoate was located above the uracil
ring of FAD (Figure S5c). The marked change in the place-
ment of the phenyl ring that is induced by the mutation
(Figure S5a) renders the mutant a catalytically active and R-
selective amine oxygenase that operates by the following
mechanism: Regarding the binding of (R)-1, dehydrogenation
can easily be performed because an a-hydrogen atom of the
substrate is directed towards the N5 atom of FAD (Fig-
ure 3b). On the other hand, for (S)-1, the a-hydrogen atom is
directed towards Tyr224. Therefore, the dehydrogenation of
(S)-1 could not be performed because the hydrogen atom was
remote from the N5 atom of FAD (Figure 3c).
was adjusted to pH 12 with NaOH (6n), and the product was
extracted four times with dichloromethane. The organic phase was
evaporated in vacuo, and the remaining product was subsequently
purified by column chromatography on silica gel (AcOEt/MeOH =
1:1). (S)-1 was isolated with 99% ee and in 64.5% yield (0.156 g,
1.29 mmol); optical rotations were measured on a ATAGO AP-300
26
automatic polarimeter (Atago Co., Tokyo, Japan). ½aꢁD ¼ꢀ29.48 (c =
20
1.02, MeOH; Ref. [12]), ½aꢁD ¼ꢀ25.78 (c = 0.98, MeOH). 1H NMR
(CDCl3, 400 MHz): d = 1.29 (d, 3H, J = 6.6 Hz), 1.61 (s, 2H), 4.01 (q,
1H, J = 6.6 Hz), 7.11–7.23 ppm (m, 5H). MS (microTOF): m/z calcd
for C8H12N1 [M+H]+: 122.0964; found: 122.0992.
Received: October 9, 2013
Revised: December 4, 2013
Published online: March 18, 2014
In conclusion, we have demonstrated that an engineered
pkDAO enzyme catalyzes the oxidation of several R-config-
ured amines to their corresponding imines with high stereo-
selectivity. This transformation could not be performed with
wild-type pkDAO. Amino acid oxidases had previously not
been described to catalyze the oxidation of amines, not even
l-amino acid oxidases, which belong to the AOx protein
family. On the other hand, the actions of AOx on amino acids
have also not been examined to date. In spite of the different
substrate specificities of the amino acid oxidase and AOx,
a single point mutation (R283G) in pkDAO led to a marked
change in the properties of the d-amino acid oxidase to give
an amine oxidase, and the mutant Y228L/R283G showed
improved catalytic activity towards (R)-1. The crystal struc-
ture of the mutant enzyme revealed the binding of (R)-1 to
the active site. The phenyl group of (R)-1 may fit into a new
hydrophobic cavity that is created by the mutation, so that the
a-hydrogen atom of (R)-1 points towards the N5 atom of
FAD. We demonstrated that enantiopure (S)-1 could be
synthesized from racemic 1 by a deracemization process using
an engineered enzyme that selectively oxidizes R-configured
amines in the presence of a chemical reductant. This is the
first study to identify a novel tailor-made flavin-containing R-
stereoselective amine oxidase that is applicable to the
production of chiral S amines by deracemization.
Keywords: amine oxidases · biocatalysis · chiral amines ·
directed evolution · oxidation
.
[1] a) M. T. Reetz, K. Schimossek, Chimia 1996, 50, 668 – 669;
b) Y. K. Choi, M. J. Kim, Y. Ahn, M. J. Kim, Org. Lett. 2001, 3,
4099 – 4101; c) D. Koszelewski, K. Tauber, K. Faber, W. Kroutil,
Trends Biotechnol. 2010, 28, 324 – 332.
[2] a) M. Alexeeva, A. Enright, M. J. Dawson, M. Mahmoudian,
N. J. Turner, Angew. Chem. 2002, 114, 3309 – 3312; Angew.
Chem. Int. Ed. 2002, 41, 3177 – 3180; b) M. Alexeeva, R. Carr,
N. J. Turner, Org. Biomol. Chem. 2003, 1, 4133 – 4137; c) H.
Leisch, S. Grosse, H. Iwaki, Y. Hasegawa, P. C. K. Lau, Can. J.
Chem. 2012, 90, 39 – 45; d) G. Li, J. Ren, H. Iwaki, D. Zhang, Y.
Hasegawa, Q. Wu, J. Feng, P. C. K. Lau, D. Zhu, Appl. Microbiol.
Biotechnol. 2014, 98, 1681 – 1689; e) D. Ghislieri, A. P. Green, M.
Pontini, S. C. Willies, L. Rowles, A. Frank, G. Grogan, N. J.
Turner, J. Am. Chem. Soc. 2013, 135, 10863 – 10869; f) D.
Ghislieri, D. Houghton, A. P. Green, S. C. Willies, N. J. Turner,
ACS Catal. 2013, 3, 2869 – 2872.
[3] M. Mure, S. A. Mills, J. P. Klinman, Biochemistry 2002, 41, 9269 –
9278.
[4] P. F. Fitzpatrick, Arch. Biochem. Biophys. 2010, 493, 13 – 25.
[5] a) C. Binda, A. Mattevi, D. E. Edmondson, J. Biol. Chem. 2002,
277, 23973 – 23976; b) P. Pietrangeli, R. Federico, B. Mondovi, L.
Morpurgo, J. Inorg. Biochem. 2007, 101, 997 – 1004.
[6] a) V. Kçhler, Y. M. Wilson, M. Durrenberger, D. Ghislieri, E.
Churakova, T. Quinto, L. Knorr, D. Haussinger, F. Hollmann,
N. J. Turner, T. R. Ward, Nat. Chem. 2013, 5, 93 – 99; b) M.
Dꢁrrenberger, T. Heinisch, Y. M. Wilson, T. Rossel, E. Nogueira,
L. Knorr, A. Mutschler, K. Kersten, M. J. Zimbron, J. Pierron, T.
Schirmer, T. R. Ward, Angew. Chem. 2011, 123, 3082 – 3085;
Angew. Chem. Int. Ed. 2011, 50, 3026 – 3029.
[7] A. Hacisalihoglu, A. Jongejan, J. A. Jongejan, J. A. Duine, J.
Mol. Catal. B 2000, 11, 81 – 88.
[8] C. Setoyama, R. Miura, Y. Nishina, K. Shiga, H. Mizutani, I.
Miyahara, K. Hirotsu, J. Biochem. 1996, 119, 1114 – 1117.
[9] V. I. Tishkov, S. V. Khoronenkova, Biokhimiya (Moscow) Engl.
Transl. 2005, 70, 51 – 67.
Experimental Section
Enzyme assay: The oxidase activity was routinely assayed at 308C by
measuring the formation of a quinone pigment and following
absorbance at 505 nm with an absorption spectrometer. The reaction
mixture contained a K2HPO4/KH2PO4 buffer (KPB; 100 mm, pH 8.0),
4-aminoantipyrine (1.5 mm), phenol (2 mm), 2 units of horseradish
peroxidase, substrate (10 mm), and enzyme.
Analytical methods: The enantiopurities of 1, 2, 3, and 4 were
analyzed by HPLC (Shimadzu, Kyoto, Japan) using a Crownpak
CR(+) column (Daicel Co., Japan) with an aqueous solution
containing HClO4 (60 mm) and MeOH (5%) as the eluent. UV
detection was performed at 210 nm.
The stereoinversion reaction of (R)-1 was performed in KPB
(100 mm; pH 8.0), with chemical reductant (20 mm), (R)-1 (1 mm),
and the enzyme.
[10] a) E. W. Hafner, D. Wellner, Proc. Natl. Acad. Sci. USA 1971, 68,
987 – 991; b) J. W. Huh, K. Yokoigawa, N. Esaki, K. Soda, J.
Ferment. Bioeng. 1992, 74, 189 – 190; c) T. M. Beard, N. J. Turner,
Chem. Commun. 2002, 246 – 247; d) F. R. Alexandre, D. P.
Pantaleone, P. P. Taylor, I. G. Fotheringham, D. J. Ager, N. J.
Turner, Tetrahedron Lett. 2002, 43, 707 – 710.
[11] A. Mattevi, M. A. Vanoni, F. Todone, M. Rizzi, A. Teplyakov, A.
Coda, M. Bolognesi, B. Curti, Proc. Natl. Acad. Sci. USA 1996,
93, 7496 – 7501.
[12] Y. Chu, Z. Shan, D. Liu, N. Sun, J. Org. Chem. 2006, 71, 3998 –
4001.
Preparative-scale synthesis of (S)-1 by deracemization: The
identity of (S)-1 that was formed by deracemization using the
purified pkDAO mutant was confirmed by its isolation. The reaction
mixture (400 mL) contained KPB buffer (40 mmol; pH 8.0), NaBH4
(40 mmol), (R/S)-1 (2.0 mmol; 0.24 g), and purified enzyme (300 U).
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