Preparation of (R)-amines from racemic amines with an (S)-amine transaminase from Bacillus megaterium

Article abstract of DOI:10.1002/adsc.200800084

Screening was carried out to identify strains useful for the preparation of (R)-1-cyclopropylethylamine and (R)-sec-butylamine by resolution of the racemic amines with an (S)-specific transaminase. Several Bacillus megaterium strains from our culture collection as well as several soil isolates were found to have the desired activity for the resolution of the racemic amines to give the (R)-enantiomers. Using an extract of the best strain, Bacillus megaterium SC6394, the reaction was shown to be a trans-amination requiring pyruvate as amino acceptor and pyridoxal phosphate as a cofactor. Initial batches of both amines were produced using whole cells of Bacillus megaterium SC6394. The transaminase was purified to homogeneity to obtain N-terminal as well as internal amino acid sequences. The sequences were used to design polymerase chain reaction (PCR) primers to enable cloning and expression of the trans-aminase in E. coli SC16578. In contrast to the original B. megaterium process, pH control and aeration were not required for the resolution of sec-butyl-amine and an excess of pyruvate was not consumed by the recombinant cells. The resolution of sec-butylamine (0.68M) using whole cells of E. coli SC16578 was scaled up to give (R)-sec-butylamine·1/2 H₂SO ₄ in 46.6% isolated yield with 99.2% ee. An alternative isolation procedure was also used to isolate (R)-secbutylamine as the free base.

Full text of DOI:10.1002/adsc.200800084

FULL PAPERS
DOI: 10.1002/adsc.200800084
Preparation of (R)-Amines from Racemic Amines withan
(S)-Amine Transaminase from Bacillus megaterium
Ronald L. Hanson,a,* Brian L. Davis,a Yijun Chen,a Steven L. Goldberg,a
William L. Parker,a Thomas P. Tully,a Michael A. Montana,a and Ramesh N. Patela
a
Process Research and Development, Bristol-Myers Squibb, One Squibb Drive, New Brunswick, NJ 08903, USA
Fax : (+1)-732-227-3994; e-mail: ronald.hanson@bms.com
Received: February 7, 2008; Published online: May 9, 2008
Abstract: Screening was carried out to identify used to design polymerase chain reaction (PCR) pri-
strains useful for the preparation of (R)-1-cyclopro- mers to enable cloning and expression of the trans-
pylethylamine and (R)-sec-butylamine by resolution
A
of the racemic amines with an (S)-specific transami- nal B. megaterium process, pH control and aeration
nase. Several Bacillus megaterium strains from our were not required for the resolution of sec-butyl-
culture collection as well as several soil isolates were
A
found to have the desired activity for the resolution by the recombinant cells. The resolution of sec-butyl-
of the racemic amines to give the (R)-enantiomers. amine (0.68M) using whole cells of E. coli SC16578
Using an extract of the best strain, Bacillus megateri- was scaled up to give (R)-sec-butylamine·1= H2SO4 in
2
um SC6394, the reaction was shown to be a trans- 46.6% isolated yield with 99.2% ee. An alternative
amination requiring pyruvate as amino acceptor and isolation procedure was also used to isolate (R)-sec-
pyridoxal phosphate as a cofactor. Initial batches of butylamine as the free base.
both amines were produced using whole cells of Ba-
cillus megaterium SC6394. The transaminase was pu-
rified to homogeneity to obtain N-terminal as well as Keywords: (R)-amines; amine transaminase; Bacillus
internal amino acid sequences. The sequences were megaterium; enzyme catalysis
Introduction
or the equilibrium is very favorable for completion of
the resolution,[15] and, conversely, synthesis of a chiral
Chiral amines are required for the synthesis of many amine from a prochiral ketone has an unfavorable equi-
pharmaceutically interesting compounds. A variety of librium.[16] However, the latter reaction has been accom-
enzymatic processes have been used for preparation of plished when conditions have been devised to shift the
chiral amines. Racemic amines have been resolved by equilibrium toward transamination of the ketone.[16,17]
acylation or deacylation using lipases or proteases[1,2] or Although the equilibrium for the resolution of racemic
by oxidation using amine oxidases.[3] Enantioselective amines is favorable, substrate and product inhibition of
acylation combined with chemical racemization of the transaminase have been reported[15,17–21] which required
amines[1,2,4] and enantioselective oxidation combined reaction engineering[15,18–20] or directed evolution of the
with non-selective chemical reduction of the imine enzyme[21] in order to carry out the reaction at higher
product[3] have allowed deracemization reactions giving substrate concentrations.
>50% yields in some cases. Enzymatic hydrolysis of the
We have explored enzymatic methods for the reso-
oxalamic esters of racemic amines has also been de- lution of cyclopropylethylamine and sec-butylamine.
scribed for the preparation of chiral secondary amines.[5] Preparation of (S)-sec-butylamine using acylation
w-Transaminases have provided another approach for with lipases was described in a previous publication
the resolution or synthesis of chiral amines.[6,7] These en- from this laboratory.[22] In this report we describe
zymes have been found in a variety of bacteria[8–13] and identification of an (S)-specific transaminase from Ba-
usually are specific for the S-enantiomer of the amine cillus megaterium SC6394, purification of the enzyme,
although an enzyme with (R)-specificity has also been cloning and expression of the gene, and preparation
described.[14] Generally an (S)-specific transaminase is of (R)-1-cyclopropylethylamine and (R)-sec-butyl-
used to convert a racemic amine to the (R)-enantiomer
A
and a ketone. When an a-k eto acid is the amino accept- coli expressing the recombinant transaminase.
Adv. Synth. Catal. 2008, 350, 1367 – 1375
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Ronald L. Hanson et al.
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Results and Discussion
Screening for Amine Transaminase
Screening was carried out to identify strains able to
produce (R)-1-cyclopropylethylamine by resolution of
the racemic amine. For this procedure, a minimal
medium containing 0.1% of the racemic amine as sole
nitrogen source was inoculated with various strains,
and after three or more days the broths were ana-
lyzed for the (R)- and (S)-amines. Strains from our
culture collection as well as isolates from soil samples
able to utilize the racemic amine as sole nitrogen
source were tested. All four Bacillus megaterium
Scheme 1. Resolution of 1-cyclopropylethylamine by trans-
amination.
strains (but not strains of six other Bacillus species) the addition of 0.1 mM pyridoxal phosphate. There
tested from the culture collection were found to have was almost no activity when a-ketoglutarate was sub-
the desired activity for resolution of the racemic stituted for pyruvate. After initial small batches in
amine to give the (R)-enantiomer. Bacillus megateri- shake flasks, resolution batches of 10 and 23 g were
um has been used as a source of w-amino acid trans- completed in a Braun Biostat B. Conditions were
aminase for the preparation of a variety of (R)- 0.082 M (1% w/v) amine, 0.164 M sodium pyruvate,
amines from racemic amines.[6,7,23] Isolation, cloning and 100 g/L cells with control of pH at 8 and aeration
and sequencing of the gene for the enzyme have been at 2 volumes/volume/min (vvm) as described in the
stated as an unpublished result.[7] Using the same Experimental Section. The solution yields were 45
screening technique with racemic sec-butylamine as and 43%. Cells were removed by centrifugation, and
the sole nitrogen source, the active strains from the the amine was recovered by distillation at pH 13, ti-
first screen also gave the (R)-enantiomer of this tration with H2SO4, removal of methanol-insoluble
1
amine. The most active strain, B. megaterium SC material and crystallization of the = H2SO4 salt from
2
6394, was grown in several different media with no ethanol (34% isolated yield, >99% ee).
addition of amines, to determine whether cells grown
in the absence of the amine could still express the
amine resolution activity. Several media that yielded Preparation of (R)-sec-Butylamine using Bacillus
active cells were identified.
A glucose/nutrisoy megaterium SC6394
medium described in the Experimental Section was
one of the best and was used in fermentation tanks to Following the procedure outlined above for 1-cyclo-
prepare cells for biotransformations and also as a propylethylamine, 30 g (0.137M) racemic sec-butyl-
source of enzyme for purification.
ACHTREUNG
Thirty-five fungi, 16 streptomycetes, 11 other acti-
ACHTREUNG
2
nomycetes and assorted Pseudomonas and Erwinia yield, 99.4% ee) using 300 g cells in 3 L (Scheme 2).
strains were also tested for activity. Few showed any To improve the productivity of the process, the reac-
effect, indicating that this particular transaminase ac-
tivity is not very common in microbes. The fungus Cy-
lindrocarpon radicicolor SC 2142 completely removed
both isomers. Several isolates selected from soil sam-
ples for ability to grow on racemic amine as sole ni-
trogen source selectively consumed the (S)-amine,
and one of the active soil isolates was identified as
Pseudomonas veronii from sequencing of the 16S ri-
bosomal RNA gene. No (R)-specific activity was
found during the screening.
Scheme 2. Resolution of sec-butylamine by transamination.
Preparation of (R)-1-Cyclopropylethylamine using
Bacillus megaterium SC6394
tion was tried with three inputs of 10 g (0.137M) race-
mic sec-butylamine plus 30.08 g sodium pyruvate
Using an extract of Bacillus megaterium SC6394, the (0.273M) added at 0 h, 47 h, and 73 h to give a solu-
resolution of 1-cyclopropylethylamine was shown to tion yield of 14.5 g (R)-sec-butylamine [no detectable
require pyruvate (Scheme 1) and to be enhanced by (S)-enantiomer) in a 1 L reaction using Bacillus mega-
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Adv. Synth. Catal. 2008, 350, 1367 – 1375

Preparation of (R)-Amines from Racemic Amines with an (S)-Amine Transaminase
FULL PAPERS
nal sequence: SLTVQKIN_EQVKE; internal sequen-
ces: LYTNRPLVVTR; SVLIGGVMPNCMR;
IGASLNVSR FVSTGSEAVETALNIAR;
GLSSSSLPAGAVLVS.
Cloning and Expression of Amine Transaminase
Mixed oligonucleotide primers were prepared based
on the amino acid sequences. PCR using these pri-
mers gave strong amplification of the expected size
Figure 1. Resolution of sec-butylamine by Bacillus megateri-
um SC6394. The reaction was started with 10 g sec-butyl-
amine, 30 g sodium pyruvate, and 100 g cells in 1 L, with ad-
ditional inputs of 10 g sec-butylamine and 30 g sodium pyru-
A
TA vector and sequenced. A labeled probe was pre-
pared and hybridized with B. megaterium chromoso-
mal DNA that had been digested with various restric-
tion endonucleases. A HindIII fragment of 4.2 kb
bound the probe and was considered large enough to
&
^
vate at 47 and 73 h. , ee of (R)-sec-butylamine,%;
,
sodium pyruvate, mg/mL.
terium SC6394 (100 g). The additional amine and pyr- contain the full gene. B. megaterium chromosomal
uvate were added each time the ee reached 99% DNA was digested with HindIII, fragments 3.8–4.8 kb
(Figure 1). Addition of 20 g amine with two equiva- isolated, ligated to pZero2, then transformed into E.
lents of pyruvate at the start of the reaction with the coli. Colonies were probed with the labeled PCR
same batch of cells only achieved 82% ee after 136 h. fragment. Plasmid DNA from hybridizing colonies
Bacillus megaterium SC6394 from other fermentation was prepared and the insert sequenced. The gene and
batches were not as productive, however, and after an protein sequences are shown in Figure 2. Significant
initial input of 10 g sec-butylamine per 100 g cells sequence homologies were found with aminotransfer-
could only resolve an additional 5 g input of racemic ase class III and 4-aminobutyrate transaminase pro-
amine.
teins from several organisms in public protein se-
quence databases. The closest homologies were to a
AHCTREUNG
Bacillus cereus aminotransferase (66% identity, 80%
similarity) and Rhodococcus sp RHA1 4-aminobuty-
rate transaminase (30% identity, 40% similarity).
PCR was used to amplify the gene, which was subse-
Purification of Amine Transaminase from Bacillus
megaterium SC6394
A novel (S)-selective aminotransferase was purified quently cloned into proprietary expression vector
446-fold from the extract to homogeneity by sequen- pBMS2004.[24] The construct pBMS2004-BMTA was
tial chromatography on Butyl-Sepharose, hydroxyapa- transformed into E. coli strain BL21. The expression
tite and UNO-Q, as described in the Experimental strain, designated E. coli SC16578, was grown in
Section and summarized in Table 1. The purified MT5M2 medium + kanamycin (Km), then induced
enzyme exhibited high activity for the transamination with b-isopropylthio-d-galactoside (IPTG) at 50 mM
between (S)-sec-butylamine and pyruvate to leave the or 1 mM. Samples were lysed by sonication and elec-
desired (R)-enantiomer. The pH optimum for the re- trophoresed on a 10% SDS-polyacylamide gel, then
action was 7.5. The purified protein had an estimated stained. A novel, overexpressed protein of the expect-
molecular weight of 50 kDa on SDS-PAGE and was ed molecular weight (53 kDa) was seen primarily in
partially sequenced to enable cloning and overexpres- the soluble fractions with either concentration of in-
sion of the enzyme. The partial amino acid sequences ducer. The strain was grown in a 250-L fermentation,
showed homology to known transaminases: N-termi- and enzyme production was induced with 50 mM
IPTG to give 12.3 kg cell paste with an (S)-transami-
nase activity of 239 mmoles/min/g cells (wet weight).
This was an 875-fold improvement in activity/g com-
Table 1. Purification of amine transaminase.
pared to the Bacillus megaterium cells.
Step
Protein Activity Specific Purification
[mg]
[U]
activity [fold]
[U/mg]
AHCTREUNG
Resolution with E. coli Expressing Recombinant (S)-
Transaminase
Extract
Butyl-Sephar- 40.2
ose
Hydroxyapatite 1.8
UNO Q1
850
14.43
2.95
0.017
0.073
4.4
E. coli SC16578 expressing the B. megaterium transa-
minase was evaluated in 5-mL reactions. sec-Butyl-
5.15
0.45
2.86
7.50
168
446
0.06
A
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Ronald L. Hanson et al.
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Figure 3. Resolution of sec-butylamine by Escherichia coli
SC16578. 750 g sec-butylamine were treated with 750 g
sodium pyruvate and 375 g E. coli SC16578 in a 15 L reac-
&
tion as described in the Experimental Section. , ee of (R)-
^
sec-butylamine,%; , sodium pyruvate, mg/mL.
tion is shown in Figure 3. Results at the 15-L scale
were nearly identical to results for the 1-L reaction.
In the larger batch, 750 g of racemic sec-butylamine
were resolved to give 585 g (R)-sec-butylam-
G
The rapid resolution at a high concentration of sec-
butylamine with simple reaction and work-up proce-
AHCTREUNG
dures makes this a very practical process.
(R)-sec-Butylamine was also isolated as the liquid
free base (36% yield, 99.1% ee) rather than the solid
= H2SO4 salt. An initial distillation at pH 3 removed
2
Figure 2. Gene and protein sequence for amine transami-
nase from Bacillus megaterium SC6394.
1
the 2-butanone. The amine was then distilled at
pH 11. Redistillation decreased the water content of
the amine to 2.5–2.9%. Treatment with type 3 A mo-
resolved in 1 h by the cells (50 mg/mL) but the resolu- lecular sieves further decreased water to 0.08%.
tion was incomplete with 100 mg/mL sec-butylamine
and 100 mg/mL cells. At the higher concentration the
ee reached 30% in 1 h but only 34% in 16 h, indicat- Conclusions
ing that the activity was not stable under these condi-
tions. E. coli input (25 mgmLÀ1) at half of the weight Screening identified a Bacillus megaterium strain with
of racemic sec-butylamine was sufficient to complete an (S)-transaminase able to resolve racemic amines to
the 50 mgmLÀ1 resolution in 15 h at pH 7.5 or pH 8.0. (R)-amines.Whole cells of the B. megaterium strain
In contrast to the original B. megaterium process, pH SC6394 were used to resolve 1-cyclopropylethylamine
control and aeration were not required, and an excess and sec-butylamine to the (R)-amines with >99% ee.
of pyruvate was not consumed by the cells.
The enzyme was purified, and the sequences enabled
The reaction was scaled up to 1 liter in a Braun cloning and expression of the transaminase gene in E.
Biostat. Racemic sec-butylamine at a concentration of coli, inducible by IPTG. Cells containing the recombi-
0.684 M (50 g), sodium pyruvate at a concentration of nant transaminase were used to resolve 750 g racemic
0.454 M (50 g, 1.33 equivalents), and E. coli SC16578 sec-butylamine (at a concentration of 0.684M) result-
(25 g) were stirred overnight at pH 8 and 308C to ing in 46.6% (maximum is 50%) isolated yield of (R)-
give 24.3 g (R)-sec-butylamine (48.6% solution yield, amine with >99% ee. The amine was efficiently
100% ee). The reaction was then scaled up to 15 L removed from the reaction mixture by distillation
1
and the amine was isolated by distillation and crystal- and isolated either as the = H2SO4 salt or as the free
2
1
lization as the = H2SO4 salt. The course of the reac- base.
2
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Preparation of (R)-Amines from Racemic Amines with an (S)-Amine Transaminase
FULL PAPERS
tention times were: (R)-enantiomer, 15.7 min; (S)-enantio-
mer 16.9 min.
Experimental Section
Screening
Analysis of Pyruvate
The test medium contained 0.1 M pH 7 phosphate buffer,
0.02% MgSO4, 0.001% NaCl, 0.001% FeSO4·7H2O, 0.001%
MnSO4·4H2O, 1% glycerol, and 0.1% filter-sterilized cyclo-
propylethylamine or sec-butylamine. One mL of sterile test
medium in a 15-mL, loosely capped tube was inoculated by
loop (from slants) or with one drop of liquid (from vials).
Tubes were shaken at 250 rpm, 288C for three or more days,
then a sample was removed for HPLC analysis of the amine
enantiomers.
Sodium pyruvate samples containing about 1 mg of sodium
pyruvate were diluted with 0.1 mL 1N HCl and water to
bring the volume to 1 mL. After standing to allow protein
precipitation, samples were filtered into HPLC vials. Sam-
ples were analyzed with a YMC PakPro C 15”0.46 cm 3 m
18
column. The mobile phase was a gradient of 10 to 90% ace-
tonitrile/water (0.05% trifluoroacetic acid in both) from 0 to
12 min and 10% acetonitrile/90% water (0.05% trifluoroace-
tic acid in both) from 12.01 to 15 min at a flow rate of 1 mL/
min, with detection at 220 nm, temperature 408C, and an in-
jection volume of 5 mL. The retention time for pyruvate was
2.1 min.
Analysis of 1-Cyclopropylethylamine
Samples of 4 to 40 mL containing about 40 mg amine were
mixed with 0.2 mL 50 mM Na2CO3 (adjusted to pH 9.5 with
HCl) and 0.2 mL dansyl chloride (2 mg/mL in acetonitrile)
in a microfuge tube and incubated for 30 min at room tem-
perature. The reaction was quenched by addition of 0.02 mL
2.8% NH4OH and incubated for an additional 15 min at
room temperature. For chiral analysis, the mixture was vor-
texed for 30 sec with 1 mL ethyl acetate, centrifuged briefly,
then 1 mL of the upper layer was dried at 408C under N2
and the residue was dissolved in 1 mL of the mobile phase
for the chiral column. For quantitation with a C18 column,
the mixture was diluted to 1 mL with 50% acetonitrile/50%
water.
After filtration through 0.2 micron nylon filters into
HPLC vials, samples were analyzed with a YMC PakPro
C18 15”0.46 cm 3 m column. The mobile phase was a gradi-
ent of 20 to 90% acetonitrile/water (0.05% trifluoroacetic
acid in both) from 0 to 12 min and 20% acetonitrile/80%
water (0.05% trifluoroacetic acid in both) from 12.01 to
15 min at a flow rate of 1 mL/min, with detection at 220 nm,
temperature 408C, and an injection volume of 5 mL. The re-
tention time for dansylated 1-cyclopropylethylamine was
9.9 min.
Growthof Bacillus megaterium SC6394
Bacillus megaterium SC6394 was used for the production of
cell paste containing (S)-transaminase. Four frozen vials of
SC6394 were thawed and their entire contents (1.5 mL)
were transferred to each of four 4-L flasks containing 1 liter
of SG-M0 medium. SG-M0 was composed of 2.2% Cerelose
(Corn Products), 0.5% Bacto yeast extract (Becton Dickin-
son and Co.), 0.5% Toasted Nutrisoy Flour (Archer Daniels
Midland) and 0.5% K2HPO4. These inoculum flasks were in-
cubated at 288C and 230 rpm for 29 h. The four flasks were
then pooled and transferred to a 1000-L tankemploying a
working volume of 750 liters containing the same medium
supplemented with 0.04% SAG5693 antifoam (GE Sili-
cones) and 0.04% Dow Corning AF Antifoam. Operating
parameters for the tankwere 28 8C, 300 rpm (287 m/min tip
speed), and an airflow of 750 L/min. No pH or dissolved
oxygen control was employed, and SAG 5693 antifoam was
added as needed to control foaming. The batch was harvest-
ed after 72 h by centrifugation, and the cells were washed
with 50 mM pH 7 potassium phosphate buffer. The 26.6 kg
of cell paste was stored at À708C and had a transaminase
activity of 0.273 mmoles/min/g cells.
For determination of ee, the samples were analyzed with a
ChiralpakOD 25”0.46 cm column (Daicel Chemical Indus-
tries, Ltd.). The mobile phase was 95% hexane/5% ethanol,
flow rate was 1 mL/min, detection was at 220 nm, tempera-
ture was 188C, and injection volume was 10 mL. Retention
times were: dansylated (R)-1-cyclopropylethylamine,
11.5 min; dansylated (S)-1-cyclopropylethylamine, 12.6 min.
Preparation of (R)-1-Cyclopropylethylamine
Racemic 1-cyclopropylethylamine hydrochloride (23 g, 189
mmoles) was dissolved in 184 mL water, 46 mL 10N NaOH
was added and the solution was kept overnight at room tem-
perature in a tightly capped bottle. This batch of amine ap-
peared to contain a transaminase inhibitor and the base
treatment improved reactivity. The solution was cooled on
ice, then about 40 mL concentrated HCl (11.6M) were
added to adjust the solution to pH 8. Potassium phosphate
buffer (230 mL of 1M, pH 8, diluted with water to 2012 mL)
was prepared. Bacillus megaterium SC6394 wet cell paste
(230 g cells stored at À708C) was thawed and dispersed in
1 L of the buffer with an Ultraturrax T25 homogenizer. The
cell suspension was added to a 5-L vessel for a Braun Bio-
statꢂ B, then sodium pyruvate (41.6 g, 378 mmoles) dis-
solved in 200 mL of the phosphate buffer was added. The
vessel was connected to a Braun Biostatꢂ B equipped with
pH and oxygen electrodes and the suspension was stirred at
300 rpm. The amine solution was added to the stirred sus-
pension and the remainder of the buffer was used to rinse
Quantitation and ee of sec-Butylamine
Marfeyꢁs reagent was used to give diasteromeric derivatives
that could be separated with a C18 column. A sample of
10 mL containing about 0.1 mg amine, 8 mL 1 M NaHCO3,
and 40 mL 1% w/v Marfeyꢁs reagent (N-a-[2,4-dinitrophenyl-
5-fluorophenyl]-l-alanine amide, Pierce) in acetone were
combined in a 1.5-mL microfuge tube and heated for 1 h at
408C. The samples were cooled to room temperature, then
8 mL 1N HCl and 934 mL 40% acetonitrile/water were
added, and the solutions were vortexed and filtered into
HPLC vials. Samples were analyzed with a YMC PakPro
C18 15”0.46 cm 3 m column. The mobile phase was 55%
methanol/45% water (both containing 0.05% trifluoroacetic
acid), flow rate was 1 mL/min, detection was at 340 nm,
temperature was 408C, and injection volume was 5 mL. Re-
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Ronald L. Hanson et al.
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the cells, pyruvate and amine into the vessel. The transami-
nation reaction containing 0.082 M amine and 0.164 M
sodium pyruvate in 2.3 L was carried out at 288C, 300 rpm
stirring speed, pH 8 was maintained with 10% NaOH and
8.5% H3PO4 feeds, and air was sparged from near the
bottom of the vessel at 4 L/min (2 vvm). The air exited from
the vessel through a condenser maintained at 48C. Samples
(4 mL) were assayed by dansylation and chiral HPLC, and
the reaction was stopped when the (S)-amine was no longer
detectable. After 22 h the ee was 16.2%, but the next sample
at 46 h was 100% ee. Initially O2 was 65% saturation but
dropped to 0% saturation by 22 h and remained at 0% until
the reaction was terminated after 46 h. Initially the NaOH
feed was required to maintain pH 8, but by 22 h the H3PO4
feed was required. The final concentration of sodium pyru-
vate determined by HPLC was 5.2 mg/mL.
The cells were removed by centrifugation at 16,900”g for
20 min, and then the cell pellets were washed with 120 mL
water and centrifuged again. The supernatants [2380 mL,
9.94 g (R)-amine, >99% ee, 43% solution yield] were com-
bined, adjusted to pH 13.1 with 10 M NaOH (120 g) and
mixed with 182 mL of n-butanol (to control foaming). The
mixture was distilled at atmospheric pressure, collecting
~200 mL fractions and adjusting the fractions to pH 4.0~4.5
with 10 M H2SO4 or 0.5 M H2SO4 for dilute fractions. Assay
by dansylation and reversed-phase HPLC, indicated only a
trace of product in the third fraction. The rich fractions
were combined and concentrated under vacuum, giving
28.3 g of a white solid. This was suspended with sonication
in 142 mL of methanol and filtered, washing the insoluble
material (ammonium sulfate, 3 g) with 142 mL of methanol.
The filtrate was concentrated under vacuum, chasing with
ethanol and drying to constant weight at room temperature,
to give a colorless solid; yield: 10.43 g (41.1%, uncorrected
for purity); mp 258~2608C (dec.); ee 99.4%.
added to a 5-L vessel for a Braun Biostatꢂ B. The vessel was
connected to a Braun Biostatꢂ B equipped with pH and
oxygen electrodes, and the suspension was stirred at
300 rpm. The transamination reaction containing 0.137 M
amine and 0.273 M sodium pyruvate in 3 L was carried out
at 288C, 300 rpm stirring speed, pH 8 was maintained with
10% NaOH and 8.5% H3PO4 feeds, and air was sparged
from near the bottom of the vessel at 6 L/min (2 vvm). The
air exited from the vessel through a condenser maintained
at 48C. Samples (10 mL) were assayed by derivitization with
Marfeyꢁs reagent and reverse phase HPLC, and the reaction
was stopped when the (S)-amine was not detectable. After
18 h the ee was 20.1%, but the ee of the next sample at 42 h
was 100%. Initially O2 was at 56% saturation but dropped
to 0% saturation by 18 h and remained at 0% until the reac-
tion was terminated after 44 h. Initially the NaOH feed was
required to maintain pH 8, but by 18 h the H3PO4 feed was
required. The final concentration of sodium pyruvate deter-
mined by HPLC was 4.3 mg/mL.
The cells were removed by centrifugation at 16,900”g for
20 min, and then the cell pellets were washed with 180 mL
water and centrifuged again. The supernatants were com-
bined [3140 mL, 14.7 g (R)-amine, 49% solution yield, 100%
ee], adjusted to pH 13 with 10 M NaOH (205 g) and the
amine distilled at 1 atm, collecting two 200 mL fractions.
The fractions were assayed by dansylation and HPLC of the
dansyl derivative. The second fraction contained negligible
amine and was discarded. The first fraction was redistilled,
collecting a 39 mL fraction (bp 60~1008C) and an 18 mL
fraction (bp 1008C). Dansyl/HPLC assay indicated that the
first fraction contained 99.8% of the product and the second
contained 0.2%. The combined distillate was cooled on ice
and adjusted to pH 6.6 with 11.35 mL of 10 M H2SO4. The
solution was concentrated under vacuum, giving 27 g of sem-
isolid residue. This was dissolved as much as possible in
136 mL of methanol with sonication and heating. The mix-
ture was cooled to room temperature and filtered, washing
with 136 mL of methanol. The methanol-insoluble solid,
4.06 g, was identified as ammonium sulfate by IR spectros-
copy. Concentration of the filtrate gave 19.4 g of crude sec-
Several runs with a combined input of 403 mmoles of the
racemic amine were isolated in a similar way. The combined
products (22.6 g) were refluxed in 226 mL of absolute etha-
nol, the hot solution filtered to remove a trace of ammoni-
um sulfate (94 mg) and the product crystallized from the fil-
trate (ice bath) to give (R)-1-cyclopropylethylami-
butylamine·1= H2SO4 salt, ee 99.2%, purity 97.8%.
2
ne·1= H2SO4; yield: 15.8 g; mp 250~2578C (dec.); ee 99.3%;
The solid was dissolved in 100 mL of methanol at reflux,
and 200 mL of absolute ethanol were added in 50 mL por-
tions, returning to reflux after each addition. The product
crystallized after the first addition. A total of 200 mL of sol-
vent (bp ~68 to 75.58C) was then distilled out. The mixture
was cooled (ice bath) and filtered, washing with 50 mL of
cold ethanol. The solid was dried under vacuum at room
2
1H NMR (300 MHz, DMSO-d6): d=0.23 (m, 1H), 0.37 (m,
1H), 0.47 (m, 2H), 0.86 (m, 1H), 1.19 (d, J=6.6 Hz, 3H),
2.43 (dq, J=9.0, 6.6, 6.6, 6.6 Hz, 1H), 7.74 (br, 3H); anal.
calcd. for C10H24N2O4S: C 44.74, H 9.01, N 10.44; found: C
44.51, H 9.30, N 10.50.
A second crop from 58 mL of EtOH gave 2.72 g, mp 252~
2578C (dec.), ee 99.2%. The total yield (first and second
crops) was 18.52 g, 138 mmoles, 34.2%.
temperature, giving (R)-sec-butylamine·1= H2SO4 salt, as a
2
nacreous colorless solid; yield: 17.6 g; mp 280~2958C
(dec.); ee 99.4%.
The mother liquor/wash was concentrated and the residue
recrystallized from 10 mL of MeOH-EtOAc, 1:1, giving an
Preparation of (R)-sec-Butylamine using Bacillus
megaterium SC6394
additional 0.68 g of (R)-sec-butylamine·1= H2SO4 as a color-
2
less solid, ee 99.4%, purity 97.2%. The isolated yield in the
Racemic sec-butylamine (30 g, 410 mmoles), sodium pyru-
vate (90.2 g, 820 mmoles) and 300 mL 1 M KH2PO4 were
dissolved in deionized water and brought to a final volume
of 3 L. The pH was adjusted to 7.5 with 85% H3PO4. Bacil-
lus megaterium SC6394 wet cell paste (300 g cells, stored at
À708C) was thawed and dispersed in 2 L of the substrate so-
lution with an Ultraturrax T25 homogenizer. The cell sus-
pension and the remaining 1 L substrate solution were
combined first and second crops was 36%.
Cell Transaminase Activity Assay
The activity assay contained 50 mg racemic sec-butylamine,
50 mg sodium pyruvate, 23 mL concentrated H3PO4, and 0.2
or 0.4% E. coli in 1 mL at pH 8. Samples were incubated at
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Preparation of (R)-Amines from Racemic Amines with an (S)-Amine Transaminase
Table 2. Primers for cloning transaminase.
FULL PAPERS
Oligo Sequence
Description
755
756
757
758
TIGTICARAARATHAAY
N-terminus of Bacillus megaterium (S)-aminotransferase (sense; corresponds to
LVQKIN)
GARGCIGTIGARACIGCIYT Internal amino acid of Bacillus megaterium (S)-aminotransferase (sense; corre-
sponds to EAVETAL
ARIGCIGTYTCIACIGCYTC Internal amino acid of Bacillus megaterium (S)-aminotransferase (anti-sense; corre-
sponds to EAVETAL)
ARIACIGCICCIGCIGGIA
Internal amino acid of Bacillus megaterium (S)-aminotransferase (anti-sense; corre-
sponds to LPAGAVL)
308C, 300 rpm in microfuge tubes placed horizontally on a
rotary shaker. After 30 or 60 min 0.1 mL samples from the
assay mixture were diluted with 0.4 mL water, then 10 mL of
the diluted sample was analyzed for (R)- and (S)-sec-butyl-
to a UNO Q1 (Bio-Rad) column equilibrated with 50 mM
potassium phosphate buffer pH 7.0 containing 1 mM dithio-
threitol and 50 mM NaCl. After washing with 12 mL of the
equilibration buffer, the enzyme was eluted with a 16 mL
linear gradient of salt (50 to 250 mM NaCl). The purified
protein gave a 50 kDa band on SDS-PAGE, which was sent
to the KeckProtein Sequencing Facility at Yale University
for N-terminal and internal peptide sequencing
A
crease in (S)-amine concentration was calculated using the
(R)-amine peakarea as an internal standard of 25 mg/mL.
Activity was calculated as mmoles (S)-amine consumed/min/
mg cells and was linear with time and cell concentration
under these conditions.
A similar procedure was used for Bacillus megaterium,
but with a lower substrate concentration, higher cell concen-
tration, and longer incubation time.
Cloning of Transaminase
Mixed oligonucleotide primers (Invitrogen) corresponding
to the N-terminal and two internal amino acid sequences
are shown in Table 2. Oligo pairs 755+757 and 756+758
were tried with the FailSafe series of PCR buffer (Epicentre
Technologies) and Bacillus megaterium chromosomal DNA
as template. The PCR (10 mL final volume) was carried out
in a Hybaid PCR Express thermocycler with the following
parameters: 1 min at 948C (1 cycle); 30 s at 948C, 30 s at
508C, 1 min at 728C (30 cycles); 5 min at 728C.
Activity Assay for Enzyme Purification
The assay solution contained 50 mM Tris-HCl buffer pH 7.5,
1 mg/mL sec-butylamine, 100 mM sodium pyruvate and
0.1 mM pyridoxal 5’-phosphate. The reaction was initiated
by addition of 0.1–10 mg enzyme, and continued at 288C,
200 rpm for 2 h. The reaction was terminated by adding
0.5 mL ethanol to the mixture. After mixing and centrifuga-
tion for 5 min, 10 mL of the supernatant were analyzed for
(R)- and (S)-sec-butylamine by the Marfey procedure as de-
scribed above. One unit of transaminase activity is defined
as one mmole per minute (S)-sec-butylamine consumed.
A prominent fragment of the expected size (~580 base
pairs based on comparison to an aminotransferase from Ral-
stonia solanacearum) was seen using oligos 756+758 upon
electrophoresis on a 1.0% TAE agarose gel in FailSafe buf-
fers “B” through “L”. This fragment was excised from the
gel and purified using the Qiagen QIAquickGel Extraction
kit, then ligated to vector pTOPO-TA (Invitrogen) using the
manufacturerꢁs protocol. Two mL of the mixture were trans-
formed into chemically-competent E. coli TOP10 cells (Invi-
trogen). SOC medium was immediately added (0.25 mL;
SOC=0.5% yeast extract, 2% tryptone, 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glu-
cose per liter), and the cells incubated in a shaker for 1 h at
378C and 225 rpm. Colonies containing recombinant plas-
mids were selected on LB agar plates containing 50 mgmLÀ1
kanamycin sulfate (Sigma Chemicals, St. Louis, MO). Seven
KmR colonies were tested for presence of the BMTA gene
by colony PCR with oligos 756+758 using FailSafe buffer
“D” and the above thermocycling protocol. Following elec-
trophoresis, all colonies supported amplification of the
580 bp fragment. Plasmid DNA from four positive colonies
was prepared and digested with restriction endonuclease
EcoRI. Three of the four samples released the 580 bp PCR
fragment and these were submitted for DNA sequence anal-
ysis. BLAST2 homology search indicated significant identity
(34%) of the PCR fragment with known 4-aminobutyrate
transaminases.
Purification of Amine Transaminase
The transaminase was purified to homogeneity through
three chromatographic steps. The crude extract (100 mL)
was prepared by microfluidization and centrifugation, made
up to 1 M ammonium sulfate and loaded onto a 20-mL
Butyl Sepharose column equilibrated with 50 mM potassium
phosphate buffer pH 7.0 containing 1 mM dithiothreitol and
1 M ammonium sulfate. After washing the column with
100 mL of the equilibration buffer, the enzyme was eluted
with a 100 mL linear gradient of ammonium sulfate from
1 M to 0 M The active fractions (30 mL) were pooled, con-
centrated and desalted by membrane centrifugation to
2 mL, then applied at flow rate of 1 mL/min to a hydroxy-
apatite column (CHT5–1 Bio-Scale, Bio-Rad) pre-equili-
brated with 50 mM potassium phosphate buffer pH 7.0.
After washing with 15 mL of the equilibration buffer, the
enzyme was eluted with a 25 mL linear gradient of phos-
phate (50 to 350 mM potassium phosphate at pH 7.0). A 3-
mL sample of the activity peakfrom the hydroxyapatite
column was concentrated to 2 mL and desalted, then applied
Adv. Synth. Catal. 2008, 350, 1367 – 1375
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Ronald L. Hanson et al.
FULL PAPERS
mid DNA was prepared using the Invitrogen Concert Plas-
mid kit and tested for presence of insert by PCR using Z-
Taq reagents (Takara), Oligos 756+758, and 1 mL of DNA
per reaction. Fourteen samples supported amplification of
the 580 bp fragment. Four of these were digested with Hin-
dIII and found to contain a ca. 5.0 kb insert. The unknown
portion of the BMTA gene sequence was elucidated using
primers based on the previously established PCR fragment
sequence (GeneWiz, South Plainfield, NJ).
Identification of the Complete Bacillus megaterium
Transaminase (BMTA) Gene
A probe specific for the BMTA gene was prepared. For la-
beling, the PCR DIG Probe Synthesis kit (Roche Biochemi-
cals) was used. The reaction mix contained PCR buffer,
DIG dNTP mix (dATP, dCTP, dGTP, and dUTP-digoxige-
nin), 0.4 nM each oligonucleotide, 2.5 U Expand DNA poly-
merase, and 10 ng of pTOPO-TA-756–758 plasmid DNA.
Thermocycling conditions were as described above.
A sample (5 mL) of the amplified DNA was electrophor-
esed on a 1.0% agarose gel in TAE buffer for 1 h at 100 V.
A fragment with a higher molecular weight than unlabeled
control (due to incorporation of dUTP-digoxigenin) was
seen. Ca. 3 mg of B. megaterium chromosomal DNA were di-
gested with a series of restriction endonucleases: Apa I,
BamHI, BglII, EcoRI, EcoRV, HindIII, KpnI, NotI, PstI,
and SpeI. They were electrophoresed for 18 h at 20 V and
transferred to a nylon filter using the VacuGene unit (Amer-
sham Biosciences). The digoxigenin-labeled probe (5 mL)
was hybridized to the filter in EasyHyb buffer (Roche Bio-
chemicals) for 18 hr at 428C, then processed according to
the manufacturerꢁs recommended protocol. Stringent wash
conditions were 0.5X SSC/0.1% SDS, 658C. Upon washing
and detection, strong hybridization could be seen in all
lanes of the filter. A HindIII fragment of approximately
4200 bp was chosen for further work.
Twenty mg of chromosomal DNA were cleaved with Hin-
dIII using 100 U enzyme for 2 h at 378C. The entire sample
was electrophoresed on a 0.8% TAE agarose gel and the
region from 3800 to 4800 bp excised. DNA was purified
using the QIAquickGel Isolation ikt and a sample was
tested for its ability to amplify a 580 bp PCR fragment using
oligos 756+758 (confirmed). The DNA sample was ligated
to vector pZero2 that had been digested with HindIII and
transformed into E. coli DH10B electrocompetent cells. Ali-
quots of the transformed cells (25, 100, 250 and 500 mL)
were spread onto 132 mm nylon filters placed on top of LB
Km agar plates and incubated at 378C for 20 h. The filter
that received 25 mL of the transformed cells had ca. 10,000
colonies. They were replicated onto two fresh filters that
were placed on top of LB Km agar medium. The filters
were incubated at 378C for 8 h. Colonies were lysed in situ
by placing the filters on a piece of Whatman 3MM paper
(Whatman International, Maidstone, UK) saturated with
0.5 M NaOH for 5 min. The filters were dried for 5 min on
Whatman paper, then neutralized on 3MM paper soaked in
1.0 M Tris-HCl, pH 7.5 for 2 min, and dried for 2 min. Mem-
branes were placed on top of 3MM paper saturated with
1.0 M Tris-HCl, pH7.0/1.5 M NaCl for 10 min. DNA was
cross-linked to the filters by exposure to ultraviolet light in
a Stratagene UV Stratalinker 2400 set to “auto crosslink”
mode (Stratagene, La Jolla, CA). Cell debris was removed
from the membranes by immersing in 3X SSC/0.1% SDS
and wiping the surface with a wetted Kimwipeꢂ, then incu-
bating in the same solution heated to 658C for 3 h with agi-
tation. Filters were rinsed with dH2O and hybridized with
the BMTA-specific probe as described above.
Production of Recombinant Transaminase
Strain SC16578 [E. coli BL21(pBMS2004-BMTA)] was used
to produce B. megaterium SC6394 S-transaminase. Two
frozen vials of SC16578 were thawed and the contents
(1.5 mL) were transferred to two 500 mL flasks containing
100 mL of MT5M0 medium. MT5M0 was composed of
4.0% glycerol, 2.0% Yeastamin 95 (Tate & Lyle), 0.6%
Na2HPO4, 0.3% KH2PO4, 0.125% ammonium sulfate, and
0.005% kanamycin sulfate (added post-autoclaving from a
filter-sterilized solution). The flasks were incubated at 308C
and 250 rpm for ca. 24 h. For the next inoculum stage, 5 mL
were transferred to each of four 4 L flasks containing 1 L of
the same medium. These second-stage flasks were incubated
at 308C and 230 rpm for ca. 20 h (OD600 ca. 7.5 AU/cm).
The entire contents of the four flasks were then pooled and
used to inoculate a 380 L Braun fermentor containing 250 L
of MT5M2 medium, yielding a starting OD600 of ca. 0.1 AU/
cm. MT5M2 medium was composed of 4% glycerol, 1.85%
Tastone 154 (Sensient Bionutrients), 2% Hy-Pea (Quest In-
ternational), 0.125% (NH4)2SO4, 0.6% Na2HPO4, 0.04%
UCON LB625 antifoam (Dow Chemical) and 0.005% kana-
mycin sulfate (prepared separately, filter-sterilized, and
added prior to inoculation). Operating parameters for the
tankwere 30 8C, 300 rpm (192 m/min tip speed), and an air-
flow of 250 L/min. No pH or dissolved oxygen control was
employed, and Ucon LB625 antifoam was added as needed
to control foaming.
At an OD600 of ca. 0.8 to 1.2 AU/cm, a filter-sterilized
IPTG solution was added to a final concentration of 50 mM.
The run was completed after 21 h, at which time OD600 had
peaked at 41 AU/cm. The whole broth was cooled to 4–
108C and cells were recovered by centrifugation, followed
by washing with 50 mM pH 7 potassium phosphate buffer.
A total of 12.3 kg of cell paste was recovered with transami-
nase activity of 239 mmoles/min/g cells.
Preparation of (R)-sec-Butylamine using E. coli
SC16578
Potassium phosphate buffer (2 L of 10 mM, pH 7) was
added to 375 g frozen E. coli SC16578. After thawing, the
cells were dispersed with an Ultraturrax homogenizer.
Water (10 L) was added to
a jacketed 20-L reactor,
equipped with a stirrer and pH electrode. Stirring was start-
ed at 100 rpm, 258C. Concentrated H3PO4 (345 mL, 5.07
moles) was added to the reactor followed by racemic sec-bu-
tylamine (750 g, 10.25 moles) and stirring was continued.
The pH was adjusted to 8.2 with about 20 mL concentrated
H3PO4 and the solution allowed to cool to 308C. Sodium
pyruvate (750 g, 6.82 moles) was added to the reactor fol-
lowed by the E. coli suspension. The pH was adjusted to 8.0
with concentrated H3PO4 and the final volume was brought
Approximately fifty strongly hybridizing colonies were
obtained, from which 36 were removed from the master
plate and inoculated into 1 mL TB (Terrific Broth)+Km
liquid medium and grown at 378C for 22 h, 300 rpm. Plas-
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Adv. Synth. Catal. 2008, 350, 1367 – 1375

Preparation of (R)-Amines from Racemic Amines with an (S)-Amine Transaminase
FULL PAPERS
to 15 L with water. The reaction was run at 308C, 100 rpm,
pH 8. No further adjustment or control of pH was necessary.
The reaction was continued until the ee was >99% (23 h).
The mixture was adjusted to pH 13 with 50% (19N)
NaOH (1300 g). Dow Corning antifoam (100 mL) was
added and the jacket temperature was increased to 1308C to
distill at 1 atm. SAG 5693 antifoam (100 mL) was added as
needed to control foaming. Distillate was collected until the
temperature was stable at ~1008C. Additional 500 mL frac-
tions were collected, titrating each with 1N sulfuric acid to
pH 4~6 until the quantity of sec-butylamine in the distillate
was insignificant. All distillate fractions containing signifi-
cant sec-butylamine were combined, cooled in an ice bath
and titrated to pH 6.1 with 30% (7.45N) sulfuric acid at
such a rate that the temperature remained below 308C. The
neutralized distillate was concentrated under vacuum,
adding n-BuOH to remove water azeotropically. When the
water level in the distillate was 0.4% or lower, the mixture
was filtered and the cake washed with 500 mL of n-BuOH.
The cake was dried under vacuum at 408C giving 585 g of
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2
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Adv. Synth. Catal. 2008, 350, 1367 – 1375
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