Enzymatic preparation of an (S)-amino acid from a racemic amino acid

Article abstract of DOI:10.1021/op1001534

The (S)-amino acid, (S)-2-amino-3-(6-o-tolylpyridin-3-yl)propanoic acid (3), is a key intermediate needed for synthesis of an antidiabetic drug candidate. Three enzymatic routes to 3 were explored. (S)-Amino acid 3 could be prepared in 73% isolated yield

Full text of DOI:10.1021/op1001534

Organic Process Research & Development 2011, 15, 241–248
Enzymatic Preparation of an (S)-Amino Acid from a Racemic Amino Acid
Yijun Chen, Steven L. Goldberg, Ronald L. Hanson, William L. Parker, Iqbal Gill, Thomas P. Tully, Michael A. Montana,
Animesh Goswami, and Ramesh N. Patel*
Process Research and DeVelopment, Bristol-Myers Squibb, One Squibb DriVe, New Brunswick, New Jersey 08903, U.S.A.
Abstract:
genases,11,12 and dynamic resolution.13-16 The (S)-amino acid,
(S)-2-amino-3-(6-o-tolylpyridin-3-yl)propanoic acid (3), is a key
intermediate needed for synthesis of glucagon-like peptide-1
(GLP-1) mimics or GLP-1 receptor modulators potentially
useful for the treatment of type II diabetes.17-22
We have previously used (S)-amino acid dehydrogenases
to convert keto acids to the corresponding unnatural (S)-amino
acids,23-26 which were used in the synthetic routes to several
pharmaceutical candidate compounds. In this report we describe
the conversion of the racemic amino acid 1 to (S)-amino acid
The (S)-amino acid, (S)-2-amino-3-(6-o-tolylpyridin-3-yl)propanoic
acid (3), is a key intermediate needed for synthesis of an antidia-
betic drug candidate. Three enzymatic routes to 3 were explored.
(S)-Amino acid 3 could be prepared in 73% isolated yield with
99.9% ee from racemic amino acid 1 using (R)-amino acid oxidase
from Trigonopsis Wariabilis expressed in Escherichia coli in com-
bination with an (S)-aminotransferase using (S)-aspartate as amino
donor. The (S)-aminotransferase was purified from a soil organism
identified as Burkholderia sp. and cloned and expressed in E. coli.
(S)-Amino acid 3 with 100% ee was also prepared in 68% solution
yield and 54% isolated yield from 1 using recombinant (R)-amino
acid oxidase from T. Wariabilis and an (S)-amino acid dehydroge-
nase from Sporosarcina ureae. The cofactor NADH required for
the reductive amination reaction was regenerated using formate
and formate dehydrogenase. The chemoenzymatic dynamic reso-
lution of 1 by (R)-selective oxidation with Celite-immobilized (R)-
amino acid oxidase in combination with chemical imine reduction
using borane-ammonia complex gave an 81% solution yield and
68% isolated yield of 3 with 100% ee.
(11) Bommarius, A. S. In Enzyme Catalysis in Organic Synthesis , 2nd
ed.; Drauz, K., Waldmann, H., Eds.; Wiley-VCH: Weinheim, Ger-
many, 2002; Vol. 3, pp 1047-1063.
(12) Ohshima, T.; Soda, K. In StereoselectiVe Biocatalysis, Patel, R. N.,
Ed.; Marcel Dekker: New York, 2000; pp 877-902.
(13) Schichl, D. A.; Enthaler, S.; Holla, W.; Riermeier, T.; Kragl, U.; Beller,
M. Eur. J. Org. Chem. 2008, 20, 3506.
(14) Yamaguchi, S.; Komeda, H.; Asano, Y. Appl. EnViron. Microbiol.
2007, 73, 5370.
(15) Turner, N. J. Curr. Opin. Chem. Biol. 2004, 8, 114.
(16) May, O.; Verseck, S.; Bommarius, A.; Drauz, K. Org. Process Res.
DeV. 2002, 6, 452.
(17) Haque, T. S.; Ewing, W. R.; Mapelli, C.; Lee, V. G.; Sulsky, R. B.;
Riexinger, D. J.; Martinez, R. L.; Zhu, Y.; Ruan, Z. Human glucagon-
like peptide-1 receptor modulators and their use in the treatment of
diabetes and related conditions. PCT Int. Appl. WO 2007082264 A2,
2007.
Introduction
(18) Qian, F.; Ewing, W. R.; Mapelli, C.; Riexinger, D. J.; Lee, V. G.;
Sulsky, R. B.; Zhu, Y.; Haque, T. S.; Martinez, R. L.; Naringrekar,
V.; Ni, N.; Burton, L. S. Sustained release GLP-1 receptor modulators.
U.S. Pat. Appl. US 2007099835 A1, 2007.
(19) Ewing, W. R.; Mapelli, C.; Riexinger, D. J.; Lee, V. G.; Sulsky, R. B.;
Zhu, Y. N-terminally modified GLP-1 receptor modulators. PCT Int.
Appl. WO 2006127948 A2, 2006.
(20) Ewing, W. R.; Mapelli, C.; Sulsky, R. B.; Haque, T. S.; Lee, V. G.;
Riexinger, D. J.; Martinez, R. L.; Zhu, Y. Human glucagon-like-
peptide-1 modulators and their use in the treatment of diabetes and
related conditions. PCT Int. Appl. WO 2006014287 A1, 2006.
(21) Natarajan, S. I.; Mapelli, C.; Bastos, M. M.; Bernatowicz, M.; Lee,
V.; Ewing, W. R. Human glucagon-like-peptide-1 mimics and their
use in the treatment of diabetes and related conditions. U.S. Pat. Appl.
Publ. US 20040127423 A1, 2004.
(S)-Amino acids are useful intermediates for the synthesis
of many pharmaceuticals.1,2 Several enzymatic approaches have
been applied for their preparation, including (S)-hydantoinases
combined with (S)-carbamoylases or HNO2,3,4 (S)-acylases,5,6
(S)-amidases,7,8 (S)-transaminases,9,10 (S)-amino acid dehydro-
* To whom correspondence should be addressed. Present address: SLRP
Associates, Bridgewater, NJ 08807. Telephone: (908)-725-5738. E-mail:
rameshpatelphd@yahoo.com.
(1) Patel, R. N. Coord. Chem. ReV. 2008, 252, 659.
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(3) Rusnak-Mueller, M.; May, O.; Hermsen, P.-J.; Straatmann, H. M. M. G.;
Skranc, W.; Boesten, W. H. J.; Heemskerk, D.; DeLange, B.
Preparation of chiral R -amino acids using Escherichia coli expressing
hydantoinase L-carbamoylase, and hydantoin racemase genes. PCT Int.
Appl. WO 2008067981, 2008.
(22) Natarajan, S. I.; Bastos, M. M.; Bernatowicz, M. S.; Mapelli, C.; Lee,
V.; Ewing, W. R. Preparation of human glucagon-like-peptide-1
mimics and their use in the treatment of diabetes and related conditions.
PCT Int. Appl. WO 2003033671 A2, 2003.
(23) Hanson, R. L.; Singh, J.; Kissick, T. P.; Patel, R. N.; Szarka, L. J.;
Mueller, R. H. Bioorg. Chem. 1990, 18, 116.
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Cazzulino, D. L.; Zannella, V.; Montana, M. A.; Nanduri, V. B.;
Schwarz, S. R.; Eiring, R. F.; Durand, S. C.; Wasylyk, J. M.; Parker,
W. L.; Liu, M. S.; Okuniewicz, F. J.; Chen, B.-C.; Harris, J. C.; Natalie,
K. J.; Ramig, K.; Swaminathan, S.; Rosso, V. W.; Pack, S. K.; Lotz,
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10.1021/op1001534  2011 American Chemical Society
Published on Web 08/09/2010
Vol. 15, No. 1, 2011 / Organic Process Research & Development
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Scheme 1. Conversion of racemic amino acid 1 to (S)-amino acid 3 using (R)-amino acid oxidase and (S)-amino acid
dehydrogenase
3 using a recombinant (R)-amino acid oxidase from Trigonopsis
Variabilis immobilized on Celite followed by an (S)-amino acid
dehydrogenase from Sporosarcina ureae. The larger scale
preparation of amino acid 3 from racemic amino acid 1 using
the (R)-amino acid oxidase cloned from T. Variabilis and an
(S)-aminotransferase cloned from Burkholderia sp., each sepa-
rately expressed in Escherichia coli is also reported. The
chemoenzymatic dynamic resolution of racemic amino acid 1
was also examined using (R)-selective oxidation with Celite-
immobilized (R)-amino acid oxidase in combination with
chemical imine reduction.
The reductive amination reaction was initiated using (S)-amino
acid dehydrogenase after 7.5 h and was continued for 19 h until
the keto acid peak was not detected by HPLC. Under the
conditions of the reductive amination reaction, the oxidase was
inhibited, so a two-step procedure was needed. The solution
yield of (S)-amino acid 3 was 68% with >99% ee and the
isolated yield was 1.06 g, 54% overall. A phenylalanine
dehydrogenase of known sequence from S. ureae was cloned
by polymerase chain reaction (PCR) and expressed in E. coli
but was not effective for this transformation, although it was
shown to be active for reductive amination of phenylpyruvate.
Conversion of Racemic Amino Acid 1 to (S)-Amino Acid
3 Using (R)-Amino Acid Oxidase and (S)-Aminotransferase.
A similar approach was used for deracemization of amino acid
1 using (R)-amino acid oxidase combined with an (S)-tran-
saminase instead of an (S)-amino acid dehydrogenase (Scheme
2). This procedure had the advantage that both enzymes could
be added at the start of reaction. A bacterial strain producing a
suitable transaminase was isolated from a soil sample and
identified as a Burkholderia sp. by comparison of its 16S rRNA
gene to publicly available sequences. The (S)-transaminase was
purified to homogeneity from extracts of this strain by a four-
step procedure (Table 1) as described in the Experimental
Section.
Results and Discussion
The basic strategy was to carry out deracemization of the
racemic amino acid 1 by combining an amino acid oxidase with
another enzyme or a chemical reducing agent. First, the amino
acid oxidase carries out the enantioselective oxidation of only
the (R)-amino acid leaving the (S)-amino acid 3 intact. The (R)-
amino acid is oxidized by the amino acid oxidase to form the
imine 4 bound to the enzyme which can be hydrolyzed to the
keto acid 2. The keto acid 2 is converted to the desired (S)-
amino acid 3 by an amino acid dehydrogenase or a transami-
nase. Alternatively, the imine 4 bound to the enzyme is reduced
chemically to the racemic amino acid 1 for recycling.
Although an N-terminal sequence could not be obtained, four
internal sequences of the purified protein were obtained fol-
lowing tryptic digestion. A BLAST2 homology search showed
regions of homology to adenosylmethionine-8-amino-7-ox-
ononanoate aminotransferases (AAOA) from Burkholderia
cenocepacia and Borrelia cepacia. Oligonucleotide primers
were prepared based on the corresponding codons of the amino
acids for use in PCR. The direction of the primers (i.e., sense
and/or antisense) were determined using the likely location of
the amino acid sequence within the protein as compared to
similar aminotransferases. The (S)-aminotransferase gene was
cloned and overexpressed in E. coli as described in the
Experimental Section.
Conversion of Racemic Amino Acid 1 to (S)-Amino Acid
3 Using (R)-Amino Acid Oxidase and (S)-Amino Acid
Dehydrogenase. Our initial approach for preparation of (S)-
amino acid 3 is shown in Scheme 1. Starting from racemic
amino acid 1, the aim was to oxidize the (R)-amino acid to the
corresponding keto acid 2, and then carry out reductive
amination of 2 to 3 by addition of ammonium formate, NAD+,
formate dehydrogenase and a suitable (S)-amino acid dehydro-
genase. (R)-Amino acid oxidase from T. Variabilis, cloned and
overexpressed in E. coli [E. coli BL21(pBMS1000 DAAO)]
and then immobilized on Celite, was effective for the initial
oxidation step. After screening of microbial cultures for a
suitable amino acid dehydrogenase active with keto acid 2, S.
ureae SC16048 was identified as a source of the desired enzyme
for the conversion of keto acid 2 to (S)-amino acid 3. Cell
extracts of S. ureae were used as a source of (S)-amino acid
dehydrogenase. Racemic amino acid (1.95 g) was oxidized with
the (R)-amino acid oxidase, and after 5.5 h the ee was 89%.
Fermentation processes for production of (R)-amino acid
oxidase and (S)-aminotransferase in recombinant E. coli were
developed and scaled up to 250 L. About 11-13 kg of wet
cell paste was obtained from E. coli expressing (S)-aminotrans-
ferase and 7 kg of wet cell paste was obtained from E. coli
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Scheme 2. Conversion of racemic amino acid 1 to (S)-amino acid 3 using (R)-amino acid oxidase and (S)-aminotransferase
Table 1. Purification of (S)-aminotransferase from
Burkholderia sp
Scheme 3. Conversion of racemic amino acid 1 to
(S)-amino acid 3 by dynamic resolution using (R)-amino acid
oxidase and chemical reduction of imine
total
total
protein activity
specific
activity (U/mg)
purification
(fold)
step
(mg)
345
(U)
crude extract
8.02
6.37
2.25
1.99
0.021
0.150
0.616
3.18
1
7
29
148
336
butyl-Sepharose 42.6
Red-120
UnoQ
Superdex 200
3.65
0.56
0.024 0.87
7.22
Table 2. Summary of fermentation runs of recombinant
E. coli for production of (S)-aminotransferase and (R)-amino
acid oxidase
cell yield activity
Table 3. Dynamic resolution of racemic amino acids using
(R)-amino acid oxidase and 10 equiv of NH3-BH3
strain
enzyme
(kg)
(U/g cells)
E. coli SC16541 (S)-aminotransferase
11.2
11.2
13.3
7.1
0.72
0.78
0.78
33.4
38.8
pH
yield (%)
ee (%)
5.0
5.5
6.0
6.5
7.0
7.5
8.0
70
68
78
79
76
73
67
51
82
100
100
100
100
100
E. coli SC16544 (R)-amino acid oxidase
6.8
expressing (R)-amino acid oxidase (Table 2). On the basis of
the much higher activity of cells containing (R)-amino acid
oxidase relative to that of cells containing (S)-amino acid
transaminase, a 5:1 ratio of transaminase:oxidase cells was used
to prepare a combined cell extract containing both activities.
Several batches containing 9.11 g (15 g of the monosulfate
monohydrate, corrected for potency) of 1 were run in a Braun
Biostat B 2-L reactor for the purpose of developing a procedure
for scale-up of the bioconversion. The reactor was controlled
at 30 °C, 300 rpm agitation, pH 7.5 and 1 L/min aeration.
Samples were taken hourly to analyze ee and conversion. The
aeration was stopped after about 6 h when the oxidation was
complete as indicated by an ee of (S)-amino acid >99%. The
reaction was continued for an additional 16 h without aeration
to allow for conversion of 2 to 3. The solution yield was 85%
and the ee was 99.5%. The reaction mixture was ultrafiltered
to remove cellular material and the permeate mixed with
ammonium sulfate and extracted with n-butanol. The product
was crystallized from the extract as the monosulfate monohy-
drate in 73% overall yield by adding H2SO4.
(R)-amino acid oxidase in combination with chemical reduction
of the imine (initial product of the oxidase reaction) with
borane-ammonia complex (Scheme 3) at various pH values.
Before the enzyme bound imine (4) hydrolyzes to the keto acid,
borane-ammonia reduces 4 to the racemic amino acid 1 in
this dynamic resolution process.15 A suspension of racemic
amino acid 1 in phosphate buffer with ammonium formate was
added to a mixture of immobilized (R)-amino acid oxidase and
borane-ammonia complex and the suspension stirred for 20 h.
Results using 10 equiv of borane-ammonia complex are shown
in Table 3. The maximum yield of 3, 76-79%, was obtained
at pH 6.0-7.0, with ee values reaching >99.9% at pHs 6-8.
Using 20 equiv of borane-ammonia complex did not signifi-
cantly improve the results.
Conclusion
The reaction was scaled up with 607 g (1 kg of the
monosulfate monohydrate corrected for potency) of 1 to give a
66% isolated yield of 3 as the monosulfate monohydrate, ee
99.9%.
Dynamic Resolution of Racemic Amino Acid to (S)-
Amino Acid. The chemoenzymatic dynamic resolution of
racemic amino acid 1 was examined using Celite-immobilized
Three methods were evaluated for deracemization of amino
acid 1 to (S)-amino acid 3. A recombinant (R)-amino acid
oxidase from T. Variabilis expressed in E. coli selectively
oxidized the (R)-enantiomer to keto acid 2, which was converted
to (S)-amino acid 3 by an (S)-transaminase. An (S)-transaminase
from a soil organism identified as a strain of Burkholderia sp.
was purified, cloned, and expressed in E. coli and found to be
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effective for conversion of 2 to (S)-3. The (S)-amino acid 3
was also prepared from the corresponding racemic amino acid
1 using (R)-amino acid oxidase in combination with an (S)-
amino acid dehydrogenase from S. ureae. The cofactor NADH
required for reductive amination reaction was regenerated using
NAD+, formate and formate dehydrogenase. The chemoenzy-
matic dynamic resolution of racemic amino acid 1 was also
demonstrated. (R)-selective oxidation with Celite-immobilized
(R)-amino acid oxidase in combination with chemical imine
reduction with borane-ammonia afforded (S)-amino acid 3.
Ultimately, we selected the very efficient (R)-amino acid
oxidase/(S)-transaminase process for the large-scale preparation
of 3.
was mixed with 0.5 mL methanol. After centrifugation, the
supernatant was assayed by HPLC. The conversion of (R)-
amino acid to keto acid was expressed in µmol/min/mL.
The (S)-aminotransferase assay mixture (0.5 mL) contained
0.1 mL of keto acid 2 (10 mg/mL, prepared by treating racemic
amino acid 1 with (S)-amino acid oxidase from Proteus
mirabilis cloned and expressed in E. coli and (R)-amino acid
oxidase), 0.1 mL sodium aspartate (20 mg/mL), 0.2 mL 50 mM
potassium phosphate pH 7, and 0.1 mL cell extract. The reaction
was initiated by addition of the extract and incubated at 30 °C
for 60 min. The reaction was quenched with 0.5 mL ethanol
and centrifuged to remove precipitated proteins. The supernatant
was used to determine amino acid 3 by HPLC. The formation
of (S)-amino acid 3 was expressed in µmol/min/mL.
Purification of (S)-Aminotransferase. A bacterial strain
with a suitable (S)-aminotransferase activity was isolated from
a soil sample by selection for growth on 0.1% (S)-1-(4-(2-(4-
methyl-2-phenyloxazol-5-yl)ethoxy)phenyl)ethanamine as the
sole nitrogen source. The strain was identified as a Burkholderia
species by comparison of its 16S rRNA gene sequence to
publicly available data banks. For preparation of crude cell-
free extracts, 25 g (wet weight) of cells was suspended in 300
mL of 50 mM potassium phosphate buffer pH 7.0 containing
1 mM dithiothreitol (DTT) and 25 µM pyridoxal 5′-phosphate
(PLP) (buffer A), then passed three times through a Microflu-
idizer at 12,000 psi. The disintegrated cells were centrifuged at
45,000 × g for 90 min at 4 °C to remove the cell debris. The
supernatant was collected and stored at -80 °C for subsequent
enzyme purification.
Experimental Section
Source of Racemic Amino Acid 1. Racemic amino acid 1
was prepared in the Process Research and Development
department of Bristol-Myers Squibb. Both 1 and 3 afford
crystalline monohydrate monosulfates. The calculated amino
acid content is 68.8%. All assays are in terms of the free amino
acid. Amino acid 1 was used as either the free amino acid or
the monohydrate monosulfate.
HPLC Methods. Quantitation with C18 column: Samples
were analyzed with a YMC ODS A C18 15 × 0.46 cm column.
The mobile phase was a gradient of 10 to 80% acetonitrile/
water (0.05% trifluoroacetic acid in both) from 0 to 12 min at
a flow rate of 1 mL/min, with detection at 282 nm, temperature
40 °C, and an injection volume of 10 µL. Retention times were:
amino acid 1 or 3, 5.4 min; keto acid 2, broad peak centered at
6.6 min.
(S)-Aminotransferase was purified to homogeneity by a four-
step procedure as follows. Crude extract (65 mL, 345 mg of
protein) containing 0.5 M ammonium sulfate was loaded on a
20-mL Butyl-Sepharose 4 fast flow (Sigma) column equilibrated
with buffer A containing 0.5 M ammonium sulfate. The enzyme
was eluted with 100 mL of ammonium sulfate with a linear
gradient (0.5 to 0 M) at a flow rate of 1 mL/min collecting
3-mL fractions. The active fractions (21 mL) were combined
and concentrated with a PM-30 membrane (Amicon) to
approximately 3 mL, followed by dilution to 20 mL with 10
mM potassium phosphate pH 7.0 buffer containing 1 mM DTT
and 5 mM magnesium sulfate (buffer B). The diluted solution
was applied to a Red-120 dye (Sigma) affinity column (18 mL)
equilibrated with buffer B, and the column was then washed
with 25 mL of buffer B. The flow-through and wash fractions
(45 mL) were combined and concentrated with a PM-30
membrane to approximately 2 mL. The concentrated enzyme
solution was applied to a UnoQ column (1 × 3.8 cm, Bio-
Rad) equilibrated with 25 mM potassium phosphate pH 7.0
containing 1 mM DTT and 0.1 M NaCl at flow rate of 1 mL/
min. After washing with 12 mL of the buffer, the column was
eluted with 16 mL of a linear gradient (0.1 to 0.2 M NaCl)
collecting 0.5-mL fractions. The enzyme activity was in the
unbound fractions, and the active fractions (8 mL) were
combined and concentrated to 0.5 mL. The enzyme solution
was injected at flow rate of 0.5 mL/min to a Superdex column
(1.2 × 30 cm, Pharmacia) equilibrated with 25 mM potassium
phosphate pH 7.0 buffer containing 1 mM DTT and 100 mM
NaCl. The enzyme was eluted with 23 mL of the column buffer
Direct determination of enantiomeric excess with chiral
column: The same samples were analyzed with a Chirobiotic
T 25 × 0.46 cm 5 µ column. The mobile phase was: A. water:
methanol 90: 10:05% acetic acid; B. methanol: water 90:10:
0.05% acetic acid with a gradient: 0-3 min, 10% B; 3-15
min, 10-100% B; 15-20 min, 100%B. Flow rate was 1.2 mL/
min, detection was at 215 nm, temperature was 25 °C (ambient),
and injection volume was 5 µL. Retention times were: (S)-
enantiomer 3, 14.1 min; (R)-enantiomer 14.9 min.
Determination of ee via derivatization with Marfey’s
reagent:27 Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-
5-L-alanine amide from Pierce) was used following the
procedure of the manufacturer to give diasteromeric
derivatives that could be separated with a C18 column.
Samples were analyzed with a YMC ODS A C18 15 ×
0.46 cm column. The mobile phase was a 25% acetonitrile/
75% water (both containing 0.05% trifluoroacetic acid),
flow rate was 1.5 mL/min, detection was at 340 nm,
temperature was 40 °C, and injection volume was 10 µL.
Retention times were: (S)-enantiomer, 10.5 min; (R)-
enantiomer 15.3 min.
Enzyme Activity Assays. The (R)-amino acid oxidase assay
mixture (0.5 mL) contained 50 µL of racemic amino acid 1
(10 mg/mL), 0.5 µL catalase, 0.44 mL 50 mM potassium
phosphate pH 8.0, and 10 µL cell extract. The reaction was
initiated by addition of the cell extract and incubated at 30 °C
for one hour. For amino acid quantitation, the reaction mixture
(27) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591.
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collecting 0.5-mL fractions. The active fractions (1.5 mL) were
combined and concentrated to 0.2 mL. Analysis by sodium
dodecyl sulfate acrylamide gel electrophoresis confirmed purity
of a 54 kDa protein.
A sample of the purified protein was submitted for partial
amino acid sequencing. No N-terminal sequence could be
obtained, but following tryptic digestion, four internal sequences
were acquired:
competent cells (Invitrogen). Colonies containing recombinant
plasmids were selected on LB agar plates containing 50 µg/
mL kanamycin sulfate (Sigma Chemicals, St. Louis, MO).
Approximately 15,000 colonies on a Hybond N+ membrane
were hybridized with the labeled PCR probe described above.
Approximately 100 strongly hybridizing colonies were obtained;
six were removed from the master plate, inoculated into LB-
kanamycin liquid medium, and grown at 37 °C for 24 h, 250
rpm. Plasmid DNA was isolated using the Fast Plasmid DNA
kit from Eppendorf. PCR with AAOA-specific primers gave
strong amplification of the expected 600-bp fragments. One
plasmid was chosen for further study and named “pZerO2-
AAOA.”
For rapid DNA sequencing of the insert in pZerO-AAOA,
primer sites were introduced at random using the New England
Biolabs Genome Priming System kit. The AAOA gene was
identified within the 4000-base pair insert by the presence of
sequences corresponding to those expected for the partial amino
acid sequences obtained.
1) APHATQA
2) KADGVYLWDSDGN
3) KELADAAYR
4) DEGIVER
Cloning and Expression of the Burkholderia sp. (S)-
Aminotransferase Gene. For use in polymerase chain reaction
(PCR), degenerate oligonucleotide primers were prepared based
on the corresponding codons of the amino acids. The direction
of the primers (i.e., sense and/or antisense) were determined
using the likely location of the amino acid sequence within the
protein as compared to similar aminotransferases.
Two sets of oligonucleotide pairs were used with the FailSafe
series of PCR buffer (Epicentre Technologies) and Burkholderia
sp. chromosomal DNA as template. The PCR (10 µL final
volume) was carried out in a Hybaid PCR Express thermocycler
with the following parameters:
Expression of the (S)-Aminotransferase (AAOA) Gene.
Primers were prepared for amplification of the AAOA for
ligation to expression vector pBMS2000:
corresponds to
primer name
Oligo 569
sequence
GACATATTTAAAT- SwaI/NdeI/5′ end
CATATGACTTAC- Burkholderia sp.
(direction)
stage 1
stage 2
stage 3
stage 4
stage 5
94° 1 min 94° 30 s 94° 30 s
94° 30 s 72° 5 min
CGCAACGAATCT- AAOA (sense)
GCC
55° 30 s 55°f40° 30 s 40° 30 s
72° 30 s 72° 30 s
72° 30 s
5 cycles
Oligo 570
GATTTAAATC-
CCGGGTTAC-
SwaI/SmaI/stop/3′ end
Burkholderia sp.
1 cycle
4 cycles 20 cycles
GAAATACCCAGT- AAOA gene (anti-
TGCTGGGCGG sense)
A fragment of the expected size (∼1056 bp based on
comparison to known AAOA genes) was obtained using oligos
based on partial amino acid sequences 1 and 4. The reaction
was scaled up 40-fold and the entire reaction mix electrophore-
sed on a 1.0% agarose gel for 2 h at 100 v in TAE buffer (0.04
M Trizma base, 0.02 M acetic acid, and 0.001 M EDTA, pH
8.3) containing 0.5 µg/mL ethidium bromide. The fragment was
excised from the gel and purified using a Qiagen Gel Purifica-
tion Kit.
To isolate the entire gene, Burkholderia sp. DNA was first
cleaved with a series of 9 restriction endonucleases and
transferred to Hybond N+ nylon filters under alkaline conditions
using the VacuGene vacuum blotting unit (Amersham-Phar-
macia). A digoxygenin-labeled probe was prepared using
specific primers based on the known DNA sequence of the
adenosylmethionine-8-amino-7-oxononanoate aminotransferase
(AAOA) gene from B. cenocepacia (GenBank accession no.
CP000958.1) since there was near-identity of the partial amino
acid sequences obtained from Burkholderia sp. and the B.
cenocepacia AAOA protein. Hybridization to the blotted DNA
digests was performed in EasyHyb solution (Roche) under
stringent wash conditions. A single hybridizing BglII fragment
of ∼3800 base pairs was seen. On the basis of this result, BglII-
digested Burkholderia sp. chromosomal DNA from ∼3200-4200
base pairs was isolated and ligated to pZero2 vector DNA
digested with BamHI (the overhanging nucleotides are compat-
ible with those of BglII) using the FastLink kit (Epicentre). DNA
was transformed by electroporation into E. coli DH10B
The FailSafe series of buffers were tested using oligos 569
+ 570 with pZerO2-AAOA as template for PCR. Buffer “D”
gave strong amplification of the expected ∼1450-bp fragment
and was used to scale up the reaction 20-fold. A MinElute
column (Qiagen) was used to purify the fragment after addition
of 5 vol buffer PB before loading onto the column.
The PCR fragment obtained with oligos 569 + 570 (∼2
µg) was cleaved with 10 U each NdeI and SmaI in a 40 µL
volume (37 °C, 2 h). Samples were electrophoresed and purified
after excision from the agarose gel using the MinElute kit. The
digestedPCRfragment(100ng)wasligatedto30ngNdeI-SmaI-
cut pBMS2000 using the Fast Link kit. One plasmid that was
verified to contain the correct insert was named pBMS2000-
AAOA and transformed into E. coli BL21, forming strain SC
16541.
Growth of Recombinant E. coli Expressing (S)-Ami-
notransferase. For shake flask expression work, SC 16541 was
initially grown in MT5-M2 for 20-24 h, 30 °C, 250 rpm. MT5-
M2 contained 2.0% Quest Hy-Pea, 1.85% Tastone-154, 0.6%
Na2HPO4, 0.125% (NH4)2SO4, and 4.0% glycerol, pH 7.2
presterilization. Kanamycin (Km) sulfate to 50 µg/mL (from a
50 mg/mL filter-sterilized stock in dH2O) was added after
autoclaving. The optical density at 600 nm (OD600) was recorded
and fresh medium inoculated with the culture to a starting OD600
of 0.30. The flask was incubated as described above until the
OD600 reached ∼0.8-1.0. Isopropyl-thio-ꢀ-D-galactoside (IPTG)
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245

was added from a 1 M filter-sterilized stock in dH2O to the
desired final concentration (50 µM or 1 mM), and the culture
was allowed to grow for varying lengths of time before
harvesting by centrifugation.
whole broth was harvested by centrifugation. During centrifuga-
tion, the cells were washed with 50 mM pH 7.0 potassium
phosphate buffer. The cell paste (7.2 kg) was then stored at
-70 °C until used for the next processing step. (R)-Amino acid
oxidase activity of 36 U/g of wet cells was obtained.
Fermentation Process for Production of (S)-Aminotrans-
ferase. Two frozen vials of E. coli SC16541 were thawed, and
the contents (1.5 mL) were transferred to two 500-mL flasks
containing 100 mL of MT5 medium (2.0% Yeastamin, 4.0%
glycerol, 0.6% Na2HPO4, 0.3% KH2PO4, 0.125% ammonium
sulfate, 0.0246% magnesium sulfate heptahydrate and 0.005%
Km sulfate [added postautoclaving from a filter-sterilized
solution]). The flasks were incubated at 30 °C and 250 rpm for
∼24 h; then 2 mL of the broth was used to inoculate 1 L of the
same medium in each of four 4-L flasks. The second-stage flasks
were incubated at 30 °C and 230 rpm for 18-20 h (average
OD600 5.5 AU/cm). The contents of each flask were then pooled
and used to inoculate a fermentor containing 250 L of MT5-
M2 medium with 0.04% UCON LB625 antifoam (Dow
Chemical) and 0.005% Km sulfate (prepared separately and
filter-sterilized) resulting in a starting OD600 of 0.08 to 0.1 AU/
cm. The fermentation process conditions for the tank were as
follows: 30 °C; no pH control; agitation, 300 rpm; aeration,
250 Lpm; back pressure, 10 psi; foam controlled by the addition
of UCON LB625 on demand.
At an OD600 of ∼0.8 to 1.0 U/cm (about 5.5 h after
inoculation), an IPTG solution (2.98 g in 500 mL of deionized
water, filter-sterilized) was added to yield a final concentration
of 50 µM. About 24-25 h postinoculation, the whole broth
was harvested by centrifugation. During centrifugation, the cells
were washed with 50 mM pH 7.0 potassium phosphate buffer.
The cell paste (12.5 kg) was then stored at -70C until used
for the next processing step. (S)-Aminotransferase activity of
0.72 to 0.78 U/g of wet cells was obtained.
Fermentation Process for Production of (R)-Amino Acid
Oxidase. (R)-Amino acid oxidase from T. Variabilis was cloned
and overexpressed in E. coli (E. coli BL21[pBMS1000-DAAO],
designated SC 16544) by our Technical Operations group in
Syracuse, New York. Cloning and expression of this gene has
been described previously by other groups.28,29 Two frozen vials
of SC16544 were thawed and the entire contents (1.5 mL) were
transferred to each of two 500-mL flasks containing 100 mL
of MT5 medium. The F1 stage flasks were incubated at 28 °C
and 250 rpm for 24 h. From each F1 flask, 5 mL was transferred
to two 4-L flasks containing 1 L of the same MT5 medium
(total: 4 flasks). The four F2 flasks were incubated at 28 °C
and 230 rpm for an additional 22 -24 h. The 4 L of pooled
inoculum from the F2 flasks was then transferred to a 250 L
working volume tank containing MT5-M2 medium. The
fermentation process was carried out at 30 °C with 300 rpm
agitation, 250 Lpm aeration, at 10 psi pressure. At an OD600 of
∼2.0 AU/cm, IPTG (8.93 g dissolved in 500 mL deionized
water and filter-sterilized) was added to yield a final concentra-
tion of 150 µM. This addition time corresponded to a CO2 off-
gas value of ∼0.11-0.15. After 24-25 h postinoculation, the
Preparation of S. ureae Cells Containing (S)-Amino Acid
Dehydrogenase. Growth/fermentation medium was as follows:
1.0% L-phenylalanine, 1.0% peptone, 0.5% yeast extract, 0.2%
K2HPO4, 0.1% NaCl, 0.02% MgSO4 adjusted to pH 7 with
K2HPO4 or KH2PO4. Broth (0.5 mL) from frozen vials
containing S. ureae SC16048 was used to inoculate 100 mL
medium in each of four 500-mL flasks. After incubation at 28
°C, 200 rpm for two days 200 mL of the culture was used to
inoculate 15 L medium in each of two fermentors. Fermentation
was carried out at 15 L/min airflow, 500 rpm agitation and 28
°C. No pH control was used during the fermentation. After 48 h,
cells were collected with a Sharples centrifuge, washed with
10 mM potassium phosphate buffer pH 7, then stored at -70
°C until used. 412 g cell paste was recovered from the two
tanks.
Conversion of Racemic Amino Acid 1 to (S)-Amino Acid
3 by (R)-Amino Acid Oxidase and (S)-Amino Acid Dehy-
drogenase. S. ureae SC16048 cells (20 g, stored frozen at -70
°C) were suspended in 50 mM ammonium formate, pH 8,
containing 1 mM DTT, to a volume of 100 mL using an
Ultraturrax homogenizer, and then disrupted by sonication for
3 min. The sonicated suspension was centrifuged at 27,500xg
for 20 min, and the supernatant was used as a source of (S)-
amino acid dehydrogenase. Racemic amino acid 1 (1.95 g, 7.61
mmol) was dissolved in 95 mL water, 5 mL 1 M potassium
phosphate buffer pH 7 added and the solution adjusted to pH
8. Immobilized (R)-amino acid oxidase (T. Variabilis enzyme
expressed in E. coli and immobilized on Celite, 4 g, 280 units
from Bristol-Myers Squibb Industrial Division) and catalase (0.2
mL, 7840 units from Biocatalytics) were added and the mixture
was shaken at 28 °C, 250 rpm in a 250-mL flask. After 5.5 h
the ee was 89%. After 7.5 h, ammonium formate (2.52 g, 40.0
mmol), dithiothreitol (31 mg, 0.2 mmol), NAD (66 mg, 0.1
mmol), formate dehydrogenase (20 mg, 10 units from Boe-
hringer) and extract from S. ureae SC16048 (60 mL) were
added, and the pH was adjusted to 8.0 with 1N NaOH. The
flask was incubated at 28 °C, 40 rpm, for 19 h at which time
keto acid 2 was not detected by HPLC analysis. The flask was
placed in a boiling water bath for 5 min to precipitate proteins,
cooled to room temperature, then centrifuged at 27,000 × g
for 20 min. The pellet was washed with 20 mL water and
centrifuged again. The combined supernatants contained 1.33 g
(68% solution yield) (S)-amino acid 3 with 100% ee in
176 mL.
The reaction mixture was adjusted to pH 7.0 (H2SO4),
mixed with 35 g of Na2SO4 and 3 extracted into n-butanol
(176- and 44-mL portions). The amino acid was extracted from
the combined rich organic phase with one 49-mL and two 10-
mL portions of 0.1 M H2SO4. The combined aqueous phase
was concentrated to 12 g (crystallization was abundant by 25 g),
mixed with 12 mL of EtOH, and left at room temperature to
equilibrate. Filtration and drying in Vacuo gave 1.70 g of 3 as
the monohydrate monosulfate, potency 62.1 w% (Na2SO4
(28) Komatsu, K.; Matsuda, A.; Sugiura, K. D-Amino acid oxidase, its gene
cloning and sequencing, and manufacture with Escherichia. JP
62262994 A, 1987.
(29) Lin, L.-L.; Chien, H. R; Wang, W.-C.; Hwang, T.-S.; Fu, H.-M.; Hsu,
W.-H. Enzyme Microb. Technol. 2000, 27, 482.
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contaminant), ee -99.9%. The isolated product thus contains
1.06 g (4.12 mmol) of 3 for an overall yield of 54% from 1.
Conversion of Racemic Amino Acid 1 to (S)-Amino Acid
3 with (R)-Amino Acid Oxidase and (S)-Aminotransferase.
Cell extracts were prepared from 200 g (wet weight) of
recombinant E. coli SC16541 cells containing (S)-aminotrans-
ferase and 40 g (wet weight) of recombinant E. coli SC16544
containing (R)-amino acid oxidase. Combined cells were
suspended in 760 mL of 100 mM potassium phosphate buffer,
pH 7.0, containing 5 µM pyridoxal 5′-phosphate (PLP), and
then disintegrated with a Microfluidizer at 10,000 psi in three
successive passes. To the cell extract, 5 g of 50% polyethyl-
eneimine (PEI) was added to a final concentration of 0.2% and
mixed. The PEI treated mixture was centrifuged at 15,000 × g
for 2 min at 4 °C to remove the cell debris. The supernatant
was collected and used for the bioconversion process.
Bioconversion of racemic amino acid 1 to (S)-amino
acid 3 was conducted in a 2-L reactor controlled by a
Braun Biostat B for optimization of the process. Racemic
amino acid 1 (15 g of the monohydrate monosulfate,
potency 60.72%, 0.0355 mol), sodium aspartate mono-
hydrate (25 g, 0.144 mol) and ascorbic acid (5 g, 0.028
mol) were dissolved in 50 mL of propylene glycol and
430 mL of water. The solution was adjusted to pH 7.5
with 10 N NaOH, then 2 mL of catalase (392 KU), 500
mL of cell extract containing 165 units of (R)-amino acid
oxidase and 72 units of (S)-aminotransferase and 2 mL
of antifoam SAG-5693 were added. The reaction was
carried out at 30 °C, pH 7.5, with stirring at 300 rpm,
and aeration at 1 L/min. Samples were taken every hour
to analyze ee and conversion yield. The aeration was
stopped at 6 h when the oxidation was complete (ee of
>99% by HPLC analysis). The transamination reaction was
continued for an additional 16 h without aeration, and the
reaction mixture was harvested.
The reaction mixture was ultrafiltered with a Millipore
30 kDa NMWL Biomax polyethersulfone membrane,
diafiltering with water to recover the product. The
combined permeate, 2.8 L, was concentrated in Vacuo to
0.73 L, washed with ethyl acetate (to remove antifoam
agent), mixed with 439 g of ammonium sulfate and
extracted with two 183-mL portions of n-butanol, The rich
extract was washed with two small portions of water to
remove residual ammonium sulfate. Assay at this point
indicated the extract contained 29.7 mmol of 3. The extract
was mixed with 59.5 mL of methanol and 59.5 mL of 1
M sulfuric acid (100% excess). The resulting mixture was
stirred with cooling in an ice bath for 4 h and filtered,
washing the solid with 60 mL of ice-cold methanol-water,
9:1. The solid was dried in Vacuo at room temperature,
giving 9.68 g of 3 as the monohydrate monosulfate,
potency 68.1%, ee 99.85.
30 min, followed by centrifugation with a Sharples
centrifuge at 0.4 gal/min to clarify the extract.
To a 100-L vessel containing 29 L of water, stirring at 100
rpm, were added sodium aspartate monohydrate (1.67 kg, 9.65
mol), ascorbic acid (0.33 kg, 1.87 mol), racemic amino acid 1
monosulfate monohydrate (1 kg, potency 60.7%, 2.37 mol), and
propylene glycol (3.33 kg). The solution was adjusted to pH
7.0 with 6.25 M NaOH. Subsequently, catalase (0.133 kg, 25500
KU), pyridoxal phosphate (167 mg, 0.63 mmol), clarified cell
extract (40 L), and SAG-5693 antifoam (133 g) were added to
the reactor. The reaction was carried out at 30 °C, 250 rpm,
pH 7.5 and 80 L/min aeration. Samples were taken every 2 h
to assay ee of the product and conversion. The oxidation was
complete within 2 h, and the bioconversion was complete after
25 h to give a 75% solution yield and ee >99.9% of 3.
The reaction mixture, 78 L, was ultrafiltered on a 20 ft2
tangential flow Millipore Biomax polyethersulfone membrane,
diafiltering with water. The combined permeate, 105.2 kg,
containing 412 g (1.61 mol) of 3, was concentrated in Vacuo
to 41 L and stirred with 21 kg of ammonium sulfate and 10.3
L of n-butanol. The aqueous phase, including some interfacial
precipitate, was separated, filtered, and extracted with 4 L of
n-butanol. The combined rich organic extract was stirred with
3.2 L of methanol and 3.2 L of 1 M sulfuric acid. A small
sample of the solution was withdrawn, rubbed with a glass rod
to induce crystallization, and the resulting slurry was returned
to the reactor. The mixture was stirred at room temperature for
one hour and then at 0 °C for 15 h. The product was filtered
out and washed with 3.4 L of cold methanol-water, 9:1, and
dried in Vacuo at 40 °C for 27 h, giving 580 g of 3 monosulfate
monohydrate, potency 69.5%, ee 99.9%, overall yield 66%.
Dynamic Resolution of Racemic Amino Acid 1 to (S)-
Amino Acid 3: Effect of pH. Portions of a suspension of 1,
13.5 mg of the sulfate monohydrate (0.036 mmol) per mL in
water containing 0.2 M ammonium dihydrogen phosphate and
0.1 M ammonium formate were adjusted to pH 5.0-8.0 at 0.5
pH unit intervals (NH4OH). One-mL portions of each was added
to a mixture of 50 mg of immobilized (R)-amino acid oxidase
on Celite and 11 mg of borane-ammonia complex (0.36 mmol)
and the suspension stirred at room temperature for 20 h. The
mixture was diluted with 1 mL of 0.1 M NaOH followed by
34 mL of 50 mM HCl. HPLC analysis gave the results shown
in Table 3.
Preparative Dynamic Resolution of Racemic Amino Acid
1 to (S)-Amino Acid 3. A suspension of 1.08 g of 1
monosulfate monohydrate (2.90 mmol) in 80 mL of 0.2 M
ammonium phosphate buffer, pH 6.0, containing 50 mM
ammonium formate, was adjusted to pH 6.25 (NH4OH). The
mixture was stirred and 0.92 g (29.8 mmol) of borane-ammonia
complex added followed by 4.0 g of (R)-amino acid oxidase
immobilized on Celite. The mixture was stirred at room
temperature. At 20 h the pH, 6.84, was adjusted to 6.25 with 1
M H2SO4, and this was repeated at 72 h (pH 6.81). HPLC at
this point indicated a solution yield of 81% and an ee of 100%.
To catalytically decompose the excess borane reagent, 0.1 g of
10% Pd/C was added and the mixture stirred for another 20 h.
The pH was adjusted to 4.0 with 2 M H2SO4, 5 g of activated
charcoal was added, and the mixture was filtered (Celite). The
Scale-Up of Bioconversion Process. Frozen SC16541
cells (12 kg) and SC16544 cells (2.4 kg) were thawed
and suspended in 46 L of 0.1 M phosphate buffer (pH
8.0) containing 5 µM PLP with agitation at 200 rpm. The
24% cell suspension was passed twice through a microf-
luidizer. PEI was added to 0.2% concentration, mixed for
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247

filtrate was concentrated in Vacuo to 11 mL, cooled on ice, and
filtered to give 0.73 g of 3 as the monosulfate monohydrate,
68% yield, ee 100%.
immobilized on Celite and Dr. Suo Liu for the strain of E. coli
expressing (R)-amino acid oxidase. We thank Dr. Robert
Waltermire for reviewing this manuscript and making helpful
suggestions.
Acknowledgment
We thank our Discovery Chemistry collaborator Yeheng Zhu
for the initial supplies of racemic amino acid 1 and for
transferring the chemistry to the scale-up group. We are grateful
to Dr. Michael Politino for providing (R)-amino acid oxidase
Received for review June 2, 2010.
OP1001534
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