Reductive amination of ketones with ammonium catalyzed by a newly identified Brevibacterium epidermidis strain for the synthesis of (S)-chiral amines

Article abstract of DOI:10.1016/S1872-2067(18)63108-0

The asymmetric reductive amination of achiral ketones with ammonia is a particularly attractive reaction for the synthesis of chiral amines. Although several engineered amine dehydrogenases have been developed by protein engineering for the asymmetric reductive amination of ketones, they all display (R)-stereoselectivity. To date, there is no report of an (S)-stereoselective biocatalyst for this reaction. Herein, a microorganism named Brevibacterium epidermidis ECU1015 that catalyzes the (S)-selective reductive amination of ketones with ammonium has been successfully isolated from soil. Using B. epidermidis ECU1015 as the catalyst, the asymmetric reductive amination of a set of phenylacetone derivatives was successfully carried out, yielding the corresponding (S)-chiral amines with moderate conversion and >99% enantiomeric excess.

Full text of DOI:10.1016/S1872-2067(18)63108-0

Chinese Journal of Catalysis 39 (2018) 1625–1632
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c h n j c
Article
Reductive amination of ketones with ammonium catalyzed by a
newly identified Brevibacterium epidermidis strain for the synthesis
of (S)‐chiral amines
Qing‐Hua Lia, Yuan Dongb, Fei‐Fei Chena, Lei Liua, Chun‐Xiu Lia, Jian‐He Xua, Gao‐Wei Zhenga,*
a State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
b Department of Pharmacy, 302 Hospital of People’s Liberation Army, Beijing 100039, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The asymmetric reductive amination of achiral ketones with ammonia is a particularly attractive
reaction for the synthesis of chiral amines. Although several engineered amine dehydrogenases
have been developed by protein engineering for the asymmetric reductive amination of ketones,
they all display (R)‐stereoselectivity. To date, there is no report of an (S)‐stereoselective biocatalyst
for this reaction. Herein, a microorganism named Brevibacterium epidermidis ECU1015 that cata‐
lyzes the (S)‐selective reductive amination of ketones with ammonium has been successfully iso‐
lated from soil. Using B. epidermidis ECU1015 as the catalyst, the asymmetric reductive amination of
a set of phenylacetone derivatives was successfully carried out, yielding the corresponding
(S)‐chiral amines with moderate conversion and >99% enantiomeric excess.
Received 30 April 2018
Accepted 24 May 2018
Published 5 October 2018
Keywords:
Biocatalysis
Reductive amination
Asymmetric synthesis
Prochiral ketones
Chiral amine
© 2018, Dalian Institute of Chemical Physics, Chinese Academy of Sciences.
Published by Elsevier B.V. All rights reserved.
1. Introduction
a challenge in industry [5,6]. Moreover, these transition met‐
al‐catalyzed processes are usually expensive and unsustaina‐
Optically pure amines are an important class of chiral in‐
termediates frequently used in the synthesis of active pharma‐
ceutical ingredients, fine chemicals, and agrochemicals [1,2].
For example, approximately 40% of the drugs approved by the
FDA contain one or more chiral amine moieties [3,4]. Due to the
prevalent use of chiral amines in organic synthesis, efficient
synthetic methods such as the asymmetric reductive amination
of ketones and asymmetric reduction of ketimines using transi‐
tion metal catalysts have been extensively developed [1,2].
However, the direct asymmetric reductive amination of
prochiral ketones with ammonia using transition metal cata‐
lysts, a key reaction for the synthesis of chiral amines, remains
ble, often requiring harsh reaction conditions with tedious
protection and deprotection steps.
With advances in protein engineering technology, biocata‐
lytic processes have been extensively developed as promising
alternatives to traditional chemocatalytic routes for the syn‐
thesis of chiral amines owing to their green credentials [7–14].
Numerous biocatalytic routes including (dynamic) kinetic res‐
olution of racemic amines by lipases [15–18], transaminases
[19–24], amine oxidases [25–32], and reductive aminases [33];
asymmetric reduction of imines by imine reductases (IREDs)
[34–42] and artificial IREDs [43–45]; and asymmetric amina‐
tion of ketones by IREDs [46–50], transaminases [51–56], re‐
* Corresponding author. Tel/Fax: +86‐21‐64250840; E‐mail: gaoweizheng@ecust.edu.cn
This work was supported by the National Natural Science Foundation of China (21472045, 21536004), and the National Defense Scientific and Tech‐
nological Innovation Special Zone (17‐163‐12‐ZT‐003‐055‐01).
DOI: 10.1016/S1872‐2067(18)63108‐0 | http://www.sciencedirect.com/science/journal/18722067 | Chin. J. Catal., Vol. 39, No. 10, October 2018

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Qing‐Hua Li et al. / Chinese Journal of Catalysis 39 (2018) 1625–1632
ductive aminases [57], and amine dehydrogenases (AmDHs)
[58–65] have been successfully developed for the synthesis of
chiral amines. Among them, the AmDH‐catalyzed asymmetric
reductive amination of ketones is a particularly attractive route
for the direct synthesis of chiral amines because it uses inex‐
pensive ammonia as the amino donor and generates only water
as the by‐product.
2.2. Screening of microbial strains
Different soil samples were collected from nature and en‐
riched using the following medium at 30 °C for 48 h. Enrich‐
ment medium (per liter): (S)‐α‐methylbenzylamine, 0.6 g (5
mmol); glycerol, 2.0 g; KH2PO4, 3.0 g; NaCl, 1.0 g; MgSO4·7H2O,
0.5 g; and trace elements; pH 7.0. The single strain isolated was
Recently, a wild‐type NADH‐dependent AmDH from the
thermophile Petrotoga mobilis was used for this reductive
amination reaction. However, the enzyme only exhibited activ‐
ity toward aliphatic ketoacids and not aliphatic ketones [66].
Bommarius and co‐workers developed two new AmDHs via
several rounds of protein engineering using naturally existing
amino acid dehydrogenases as scaffolds [58,59]. Subsequently,
three other engineered AmDHs, including Rhodococcus
Phe‐AmDH from Rhodococcus sp. M4 [63], EsLeu‐AmDH from
Exiguobacterium sibiricum [68], and thermostable Cal‐AmDH
from Caldalkalibacillus thermarum [65], were developed using
the same approach. These engineered AmDHs were used to
synthesize a broad set of chiral amines with excellent enanti‐
oselectivity by reductive amination of ketones with ammonia.
More importantly, two of them have been combined with alco‐
hol dehydrogenases for the synthesis of chiral amines from
inexpensive racemic alcohols through elegant hydrogen bor‐
rowing dual‐enzyme cascade reactions [67,68]. Very recently,
we successfully expanded the substrate scope of three engi‐
neered AmDHs by fine‐tuning two key residues surrounding
the substrate‐binding cavity, thus resulting in steric hindrance
for the binding of bulky substrates [69].
However, all these engineered AmDHs display
(R)‐stereoselectivity. Therefore, only R‐configuration chiral
amines can be synthesized from the reductive amination of
ketones with ammonia by these enzymes. To date, no
(S)‐stereoselective biocatalyst capable of catalyzing the reduc‐
tive amination of ketones with ammonia has been reported.
Herein, we report the isolation of microbial strains from soil
samples able to catalyze the reductive amination of ketones
using inexpensive inorganic ammonium as the amine donor for
the synthesis of (S)‐chiral amines.
initially
screened
for
the
deamination
of
(S)‐α‐methylbenzylamine. The reaction mixture for deamina‐
tion contained 5 mmol/L (S)‐α‐methylbenzylamine, wet cells
from 4 mL cultured broth, and 0.5 mL glycine‐NaOH buffer (0.2
mol/L, pH 10.0). The reaction was carried out at 30 °C and
1000 r/min for 24 h, and the products were analyzed by TLC.
The strains producing acetophenone were rapidly identified
and further screened for the reductive amination of acetophe‐
none with ammonia. The reaction mixture for amination con‐
tained 5 mM acetophenone, wet cells from 4 mL cultured broth,
1 mol/L NH4Cl, and 0.5 mL Tris–HCl buffer (0.2 mol/L, pH 8.0),
which was shaken at 30 °C for 24 h. The products extracted
from the reaction mixture were identified by GC.
2.3. Identification of the best strain
For the identification of the microorganism, the 16S rDNA
gene was amplified via the polymerase chain reaction (PCR)
with the universal primer pair AGAGTTTGATCCTGGCTCAG and
GGTTACCTTGTTACGACTT. The sequence was analyzed with
the Basic Local Alignment Search Tool (BLAST). In order to
identify the organism from the 16S rDNA gene sequence, the
phylogenetics and molecular evolutionary genetics were con‐
structed using the Clustal Omega and MEGA version 6.06 soft‐
ware. According to the 16S rDNA sequence of strain Es11, it
was identified as Brevibacterium epidermidis ECU1015.
2.4. Cultivation of B. epidermidis ECU1015
B. epidermidis ECU1015 was grown aerobically at 30 °C for
24 h in an optimized medium with the following composition
(per liter): glycerol, 10.0 g; peptone, 5.0 g; yeast extract, 5.0 g;
NaCl, 1.0 g; KH2PO4, 0.5 g; and MgSO4, 0.2 g; pH 7.0. After culti‐
vation, the cells were harvested by centrifugation (8000 r/min,
10 min) and washed twice with physiological saline.
2. Experimental
2.1. Reagents
2.5. Effects of different parameters on the B. epidermidis
ECU1015 activity toward reductive amination
Acetophenone (1a), 3,4‐dihydronaphthalen‐1(2H)‐one (1b),
1‐(4‐fluorophenyl)propan‐2‐one (pFPA) (1d), 1‐(4‐methoxy‐
phenyl)propan‐2‐one (1g), 1‐(3‐fluorophenyl)propan‐2‐one
2.5.1. Effect of inorganic ammonium concentration
(1e),
(S)‐α‐methylbenzylamine, (R)‐α‐methylbenzylamine, (S,R)‐4‐
fluoro‐α‐methylphenethylamine, (R)‐1‐(4‐methoxyphenyl)
propan‐2‐amine, (S)‐1‐(4‐methoxyphenyl)propan‐2‐amine,
1‐(2‐fluorophenyl)propan‐2‐one
(1f),
The effect of the concentration of inorganic ammonium on
the reductive amination catalyzed by B. epidermidis ECU1015
whole cells was studied at different concentrations of NH4Cl.
The reaction mixture (1 mL) contained 5 mmol/L pFPA (1d),
different concentrations of NH4Cl ranging from 0 to 2 mol/L,
100 g/L wet cells of B. epidermidis, 4% (v/v) DMSO, and potas‐
sium phosphate sulfate (KPB) (0.2 mol/L, pH 7.5). The reac‐
tions were performed at 30 °C and 1000 r/min for 24 h. The
samples were treated by addition of 100 μL NaOH (10 mol/L)
and extracted with dichloromethane (600 μL). The organic
and (S)‐1,2,3,4‐tetrahydronaphthalen‐1‐amine were purchased
from Sigma‐Aldrich (Tianjin, China). All the other chemicals
1
used were of analytical grade and commercially available. H
and 13C NMR spectra were recorded on a Bruker Avance 400
MHz spectrometer.

Qing‐Hua Li et al. / Chinese Journal of Catalysis 39 (2018) 1625–1632
1627
layer was dried over anhydrous sodium sulfate. The concentra‐
tion and enantiomeric excess (ee) of the products were deter‐
mined by GC analysis.
product were dried over anhydrous Na2SO4 and concentrated.
An ethereal solution of HCl (2 mol/L, 2 mL) was added to the
remaining product to precipitate the corresponding amine
hydrochloride. The precipitate was dried under reduced pres‐
sure to afford the corresponding amine hydrochloride.
2.5.2. Effect of pH and temperature
To study the effect of the pH on the B. epidermidis
ECU1015‐catalyzed reductive amination reaction, the experi‐
ments were performed at different initial pH values at 30 °C for
24 h. The reaction mixture included 0.5 mL whole‐cells con‐
taining 100 g/L wet cells of B. epidermidis, 5 mmol/L pFPA,
1.25 mol/L NH4Cl, 4% (v/v) DMSO, and different buffers with
pH values ranging from 6.0 to 10.0: KPB (pH 6.0 to 7.5),
Tris–HCl (pH 7.5 to 9.0), and glycine‐NaOH (pH 9.0 to 10.0).
The effect of the temperature was studied by running ex‐
periments at different temperatures ranging from 25 to 40 °C
for 24 h. The reaction mixture was composed of 100 g/L wet
cells of B. epidermidis, 5 mM pFPA, 1.25 mol/L NH4Cl, 4% (v/v)
DMSO, and 0.5 mL KPB (0.2 mol/L, pH 7.5).
2.8. GC analysis
Conversion analysis: the substrate conversion was deter‐
mined with a GC‐2014 gas chromatograph (Shimadzu, Tokyo,
Japan) equipped with an FID detector and a DB‐1701 column
(Agilent, 30 m × 0.25 mm × 0.25 μm) using N2 as the carrier gas.
n‐Dodecane was used as the internal standard. The injector and
detector temperatures were set to 250 and 280 °C, respectively.
The initial column temperature of 120 °C was held for 2 min,
raised to 150 °C at a rate of 10 °C/min and held for 1 min, then
raised to 200 °C at a rate of 20 °C/min, and finally held for 15
min.
The samples were treated by addition of 0.1 mL NaOH (10
mol/L) and then extracted with dichloromethane (600 μL). The
organic layer was dried over anhydrous sodium sulfate. The
concentration and ee of the products were determined by GC
analysis.
Enantiomeric excess analysis: The samples were first deri‐
vatized by adding pyridine (2 μL) and acetic anhydride (5 μL)
at room temperature for 30 min, and then characterized by
GC‐2014 gas chromatography (Shimadzu, Tokyo, Japan)
equipped with an FID detector and a CP‐Chiral‐DEX CB column
(Agilent, 25 m × 0.25 mm × 0.25 μm) using N2 as the carrier gas.
The injector and detector temperatures were both set to 280
°C. The initial column temperature of 70 °C was held for 2 min,
raised to 120 °C at a rate of 20 °C/min and held for 2 min, then
raised to 160 °C at a rate of 10 °C/min and held for 2 min, and
finally raised to 180 °C at a rate of 10 °C/min and held for 2
min.
2.5.3. Effect of substrate concentration
To research the effect of the substrate concentration on the
reductive amination reaction, experiments with mixtures con‐
taining 100 g/L wet cells of B. epidermidis, 1.25 mol/L NH4Cl,
4% (v/v) DMSO, pFPA at concentrations ranging from 5 to 30
mmol/L, and 0.5 mL KPB (0.2 mol/L, pH 7.5) were carried out
at 30 °C for 24 h.
3. Results and discussion
2.6. Reductive amination of selected ketones catalyzed by B.
epidermidis ECU1015 under optimized conditions
3.1. Identification of the best strain
Mixtures composed of 10 mmol/L of ketone (1a−1e), 1.25
mol/L NH4Cl, 100 g/L wet cells of B. epidermidis, 4% (v/v)
DMSO, and 0.5 mL KPB (0.2 mol/L, pH 7.5) were shaken at
1000 r/min in sealed 2 mL tubes at 30 °C for 24 h. The samples
were treated by addition of 0.1 mL NaOH (10 mol/L), and then
extracted with dichloromethane (600 μL). The organic layer
was dried over anhydrous sodium sulfate. The substrate con‐
version and product ee were determined by GC analysis.
Initially, various microorganisms were isolated from soil
samples
through
enrichment
culturing
using
(S)‐α‐methylbenzylamine ((S)‐α‐MBA) as the sole nitrogen
source. The strains showing clear formation of acetophenone
on the silica gel plates by TLC analysis were selected and fur‐
ther examined for amination activity, using acetophenone
(Scheme 1, 1a) as the model substrate in the presence of inor‐
ganic ammonium. Among them, two candidate strains were
NH2
B. epidermidis ECU1015
O
2.7. Preparation of (S)‐4‐fluoro‐α‐methylphenethylamine by B.
epidermidis‐catalyzed reductive amination
NH4Cl, pH 7.5, 30 oC
R
R
(S)-2a 2g
1a 1g
Wet cells of B. epidermidis ECU1015 (10 g), 152 mg 1d (10
mmol/L), 1.25 mol/L NH4Cl, 4% (v/v) DMSO, and 100 mL KPB
(0.2 mol/L, pH 7.5) were mixed in a 250‐mL round bottom
flask. The reaction mixture was shaken in an incubator at 30 C.
The reaction was monitored by GC. When the conversion
reached a plateau, the reaction mixture was acidified to pH 2.0
by addition of HCl (1 mol/L). The water layer was washed with
100 mL CH2Cl2 to remove any unreacted ketone, the pH ad‐
justed to 12.0–14.0 using NaOH (10 mol/L), and extracted with
100 mL CH2Cl2. The organic fractions containing the amine
O
O
O
1b
1c
1a
F
F
O
F
O
O
O
O
1d
1e
1f
1g
Scheme 1. Reductive amination of selected ketones 1a−1g catalyzed by
B. epidermidis ECU1015.

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Qing‐Hua Li et al. / Chinese Journal of Catalysis 39 (2018) 1625–1632
enzymes used for the reductive amination of achiral ketones
Table 1
such as amine dehydrogenases [66–69] display poor affinity
toward ammonia. Therefore, the supra‐stoichiometric addition
of ammonia is necessary to drive the reaction equilibrium in
the desired direction. For example, Carine Vergne‐Vaxelaire
and co‐workers identified a natural amine dehydrogenase from
Petrotoga mobilis. The enzyme catalyzed the transformation of
4‐ketopentanoic acid (10 mmol/L) to 4‐aminopentanoic acid
using 5 mol/L free ammonia as the amine donor [64]. There‐
fore, 1.25 mol/L was chosen as the optimal NH4+ concentration
for the reductive amination of pFPA by B. epidermidis.
Reductive amination of 1a and 1d with ammonia using two candidate
strains.
Substrate
Strain
Conversiona (%)
eeb (%)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
1a
1a
1d
1d
Strain Es11
Strain Fm98
Strain Es11
Strain Fm98
7.4
6.2
36.0
8.6
a Determined by GC (DB‐1701 column);
b Determined by GC (CP‐Chiral‐DEX column).
found to catalyze the reductive amination of 1a to (S)‐α‐MBA
with >99% ee, albeit with low conversion (Table 1). Moreover,
these two candidate strains could also convert pFPA (1d) into
the corresponding (S)‐4‐fluoro‐α‐methylphenethylamine with
>99% ee (Table 1). In this reaction, strain Es11 yielded a higher
conversion than strain Fm98 and was therefore chosen for
further studies. Strain Es11 was subsequently identified as
Brevibacterium epidermidis ECU1015 by 16S rDNA sequencing
and hereafter designated as B. epidermidis ECU1015.
3.3. Effect of pH and temperature on B. epidermidis‐catalyzed
reductive amination
To determine the optimal pH, the B. epidermidis‐catalyzed
reductive amination of pFPA was conducted at pH values rang‐
ing from 6.0 to 10.0. As shown in Fig. 2(a), the highest product
concentration was obtained at pH 7.5. Thus, pH 7.5 was chosen
as the optimal pH for the asymmetric reductive amination of
pFPA by B. epidermidis.
3.2. Effect of amino donors and ammonium concentration on B.
epidermidis‐catalyzed reductive amination
Similarly, the effect of temperature (25−40 C) was also
studied. The concentration of the product formed increased
with temperature from 25 to 30 C to then decrease at higher
temperatures (Fig. 2(b)). Hence, 30 C was chosen as the opti‐
mum reaction temperature for the asymmetric reductive ami‐
nation reaction catalyzed by B. epidermidis.
To choose the most suitable amino donors, we first exam‐
ined the effect of different amino compounds on the synthesis
of (S)‐pFPAm from pFPA. As shown in Fig. 1(a), using NH4Cl as
the amino donor, the reaction afforded the highest product
yield. Therefore, NH4Cl was selected as the amino donor for the
reductive amination reactions.
3.4. Effect of substrate concentration on the B.
epidermidis‐catalyzed reductive amination
Subsequently, different concentrations of NH4Cl ranging
from 0.25 to 2.0 mol/L were used for the amination of sub‐
strate pFPA to investigate the effect of the NH4+ concentration
on the reductive amination activity of B. epidermidis. As shown
in Fig. 1(b), the concentration of the product
(S)‐4‐fluoro‐α‐methylphenethylamine is strongly affected by
The substrate concentration was also found to have a sig‐
nificant effect on the asymmetric reductive amination catalyzed
by B. epidermidis. Therefore, the reductive amination reaction
was carried out for 24 h at different substrate concentrations.
As shown in Fig. 3, the concentration of the product increased
with the substrate concentration up to 10 mmol/L, after which
it decreased. Therefore, 10 mmol/L was chosen as the optimal
substrate concentration.
+
the NH4 concentration. The concentration of the product in‐
+
creased with the NH4 concentration and then decreased at
ionic concentrations greater than 1.25 mol/L. In general, the
2.0
2.5
(b)
(a)
2.0
1.5
1.0
0.5
1.5
1.0
0.5
0.0
0.0
0.5
1.0
1.5
2.0
2.5
NH4+ concentration (mol/L)
None
L-Ala NH4COOH NH4Cl (NH4)2SO4 NH4NO3
Fig. 1. Effect of amino donor (a) and NH4+ concentration (b) on reductive amination of pFPA by B. epidermidis.

Qing‐Hua Li et al. / Chinese Journal of Catalysis 39 (2018) 1625–1632
1629
2.5
2.2
1.7
1.2
0.7
0.2
KPB buffer
(b)
(a)
Tris-HCl buffer
Glycine-NaOH buffer
2.0
1.5
1.0
0.5
6
7
8
9
10
11
20
25
30
35
40
45
pH
Temperature (ºC)
Fig. 2. Effect of pH (a) and temperature (b) on reductive amination of pFPA. The reaction was performed using the conditions for the pFPAm synthe‐
sis, except that the initial pH of the reaction mixture at 30 C (a) and the reaction temperature (b) were varied as indicated in the figures.
nylacetone derivatives such as meta‐, ortho‐fluoro‐, and pa‐
3.5. Substrate scope for reductive amination by B. epidermidis
ra‐methoxy‐substituted phenylacetones 1e1g were examined.
Conversions of 45%68% were achieved in the B. epidermid‐
is‐catalyzed reductive amination of 1e1g and the corre‐
sponding chiral amines were obtained in >99% ee with
(S)‐selectivity (Table 2, entries 57). The moderate conversions
are possibly caused by poor expression of the target enzyme in
the natural host. Therefore, it should be possible to further
increase the conversion using recombinant enzymes or highly
active engineered mutants in the future.
Under the optimal reaction conditions, the asymmetric re‐
ductive amination of a range of ketones (Scheme 1, 1a−1g)
catalyzed by B. epidermidis ECU1015 was investigated, and the
results are shown in Table 2. For acetophenone 1a, B. epider‐
midis ECU1015 exhibited very low reactivity, affording only
9.7% conversion (Table 2, entry 1), while no measurable reac‐
tivity for bulky substrate 1b was observed, suggesting that the
steric hindrance of this bulky substrate severely hampers its
transformation. In addition, B. epidermidis exhibited almost no
activity toward aliphatic ketone 1c. Pleasingly, B. epidermidis
3.6. Preparation of (S)‐4‐fluoro‐α‐methylphenethylamine
ECU1015
ra‐fluorophenylacetone 1d
displayed
higher
activity
bearing
toward
more elec‐
pa‐
The reductive amination product of substrate 1d,
(S)‐4‐fluoro‐α‐methylphenethylamine, is an important chiral
building block for the synthesis of human adenosine receptor
agonists [72]. To assess the feasibility of this process, 1d was
subjected to preparative synthesis at 100 mL reaction scale.
The resulting time‐course (Fig. 4) shows that the substrate was
converted into optically pure (S)‐4‐fluoro‐α‐ methylphene‐
thylamine with 50% conversion and >99% ee within 36 h. After
a
tron‐withdrawing substituent than acetophenone 1a, whereby
53% of 1d was converted into the corresponding (S)‐amine
with >99% ee within 30 h (Table 2, entry 4), suggesting that
electron‐withdrawing substituents may activate the carbonyl
carbon of the substrates [70,71]. Subsequently, other phe‐
extraction
and
normal
work‐up,
58.3
mg
of
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
(S)‐4‐fluoro‐α‐methylphenethylamine hydrochloride was iso‐
lated in 30.6% yield and >99% ee.
Table 2
Reductive amination of ketones 1a1g by B. epidermidis ECU1015.
Time
(h)
48
Conversiona
(%)
Entry
Substrate
ee b (%)
1
2
3
4
5
6
7
1a
1b
1c
1d
1e
1f
9.7
n.m.c
n.m.
53
45
>99 (S)
n.m.
n.m.
48
48
30
30
30
30
>99 (S)
>99 (S)
>99 (S)
>99 (S)
49
68
5
10
15
20
30
Substrate concentration (mmol/L)
1g
a Determined by GC (DB‐1701 column);
b Determined by GC (CP‐Chiral‐DEX column);c No measurable activity.
Fig. 3. Effect of substrate concentration on reductive amination of pFPA
by B. epidermidis.

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Qing‐Hua Li et al. / Chinese Journal of Catalysis 39 (2018) 1625–1632
99% and yield of 30.6% after process optimization. In addition,
10
8
a range of (S)‐aryl amines were synthesized by biocatalytic
reductive amination of the corresponding ketones. Thus, B.
epidermidis ECU1015 has been demonstrated to be a very
promising biocatalyst for the asymmetric synthesis of (S)‐chiral
amines.
Substrate 1d
Chiral amine (S)-2d
6
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Graphical Abstract
Chin. J. Catal., 2018, 39: 1625–1632 doi: 10.1016/S1872‐2067(18)63108‐0
Reductive amination of ketones with ammonium catalyzed by a newly identified Brevibacterium epidermidis strain for the
synthesis of (S)‐chiral amines
Qing‐Hua Li, Yuan Dong, Fei‐Fei Chen, Lei Liu, Chun‐Xiu Li, Jian‐He Xu, Gao‐Wei Zheng *
East China University of Science and Technology; 302 Hospital of People’s Liberation Army
The new strain Brevibacterium epidermidis ECU1015 can catalyze the reductive amination of ketones using inorganic ammonium as the
amino donor to generate the corresponding (S)‐chiral amines with high enantiomeric excess (ee > 99%). A range of (S)‐aryl amines were
synthesized via biocatalytic reductive amination of ketones.

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Brevibacterium epidermidis催化酮不对称还原胺化合成(S)-手性胺
李清华a, 董 源b, 陈飞飞a, 柳 磊a, 李春秀a, 许建和a, 郑高伟a,*
a华东理工大学生物反应器工程国家重点实验室, 上海200237
b中国人民解放军第三〇二医院药学部, 北京100039
摘要: 光学纯手性胺是一类非常重要的手性化学品, 作为手性砌块和手性拆分剂广泛用于医药、农业化学品、精细化学品
等产品的合成中. 据统计, 美国FDA近年来批准的约40%药物中都含有一个或多个手性胺结构单元. 胺脱氢酶(AmDH)是
由氨基酸脱氢酶改造而来的一类催化酮不对称还原胺化的新酶, 其在手性胺的合成中展现出较强的潜力, 已引起国内外学
术界和工业界的广泛关注. 这是因为该酶能够利用廉价的无机铵为胺供体, 且具有催化效率高、原子经济性好和环境友好
等优点. 迄今为止已经有数个高效的胺脱氢酶被成功开发和报道, 但是这些通过蛋白质工程改造的胺脱氢酶均为(R)-选择
性, 因此只能合成(R)-选择性的手性胺, 遗憾的是还未见有(S)-选择性胺脱氢酶的报道. 因此, 本文主要目的是期望从自然
环境中鉴定能够不对称还原胺化酮合成(S)-手性胺的微生物, 进而从中分离得到能够以无机铵作为胺供体合成(S)-手性胺
的(S)-选择性酶.
本文首先利用苯乙胺作为唯一氮源, 从土壤中筛选能够利用苯乙胺生长的菌株, 进而利用苯乙酮作为初筛底物对得到
的菌株进行胺化能力筛选, 再利用(4-氟苯基)丙酮作为模式底物进行进一步的筛选. 幸运的是, 我们获得了能够利用无机
铵作为胺供体催化(4-氟苯基)丙酮不对称还原胺化合成(S)-4-氟-α-甲基苯乙胺的菌株, 经过16S RNA鉴定为表皮短杆菌, 命
名为B. epidermidis ECU1015.
接下来, 我们对B. epidermidis ECU1015催化的胺化反应中的关键参数如胺基供体及其最适浓度、反应温度、pH值和
底物浓度等进行了优化, 确定最佳反应条件: 胺供体为NH4Cl (1.25 mol/L), 反应温度为30 °C, KPB缓冲液(200 mmol/L, pH
7.5), 底物浓度10 mmol/L. 最后, 在最适的反应条件下, 我们对B. epidermidis ECU1015催化的底物谱进行了研究. 结果表
明, 该微生物不能催化大位阻芳香酮和链状酮的胺化, 对位阻较小的苯乙酮及(4-氟苯基)丙酮具有较好的还原胺化能力, 而
且对苯环上带有吸电子取代基的酮化合物具有更好的转化效果. 经手性分析, 所有生成的手性胺均为(S)-构型, 产品的光
学纯度均>99%.
B. epidermidis催化酮不对称胺化所形成的产物构型均为(S)-选择性, 这不同于已报道的(R)-选择性胺脱氢酶. 该菌株的
发现为(S)-选择性胺脱氢酶的进一步鉴定奠定了一定的研究基础, 相关蛋白的分离纯化工作正在进行.
关键词: 生物催化; 还原胺化; 不对称合成; 潜手性酮; 手型胺
收稿日期: 2018-04-30. 接受日期: 2018-05-24. 出版日期: 2018-10-05.
*通讯联系人. 电话/传真: (021) 64250840; 电子信箱: gaoweizheng@ecust.edu.cn
基金来源:国家自然科学基金(21472045, 21536004); 国防科技创新特区项目(17-163-12-ZT-003-055-01).
本文的电子版全文由Elsevier出版社在ScienceDirect上出版(http://www.sciencedirect.com/science/journal/18722067).