Characterization of a new nitrilase from Hoeflea phototrophica DFL-43 for a two-step one-pot synthesis of (S)-β-amino acids

Article abstract of DOI:10.1007/s00253-018-9057-7

A nitrilase from Hoeflea phototrophica DFL-43 (HpN) demonstrating excellent catalytic activity towards benzoylacetonitrile was identified from a nitrilase tool-box, which was developed previously in our laboratory for (R)-o-chloromandelic acid synthesis from o-chloromandelonitrile. The HpN was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity by nickel column affinity chromatography, and its biochemical properties were studied. The HpN was very stable at 30–40?°C, and highly active over a wide range of pH values (pH 6.0–10.0). In addition, the HpN could tolerate against several hydrophilic organic solvents. Steady-state kinetics indicated that HpN was highly active towards benzoylacetonitrile, giving a K_M of 4.2?mM and a k_cat of 170?s^?1, the latter of which is ca. fivefold higher than the highest record reported so far. A cascade reaction for the synthesis of optically pure (S)-β-phenylalanine from benzoylacetonitrile was developed by coupling HpN with an ω-transaminase from Polaromonas sp. JS666 in toluene-water biphasic reaction system using β-alanine as an amino donor. Various (S)-β-amino acids could be produced from benzoylacetonitrile derivatives with moderate to high conversions (73–99%) and excellent enantioselectivity (> 99% ee). These results are significantly advantageous over previous studies, indicating a great potential of this cascade reaction for the practical synthesis of (S)-β-phenylalanine in the future.

Full text of DOI:10.1007/s00253-018-9057-7

Applied Microbiology and Biotechnology
https://doi.org/10.1007/s00253-018-9057-7
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Characterization of a new nitrilase from Hoeflea phototrophica DFL-43
for a two-step one-pot synthesis of (S)-β-amino acids
1
1
1
Zhi-Jun Zhang
& Rui-Feng Cai & Jian-He Xu
Received: 12 March 2018 /Accepted: 27 April 2018
Springer-Verlag GmbH Germany, part of Springer Nature 2018
#
Abstract
A nitrilase from Hoeflea phototrophica DFL-43 (HpN) demonstrating excellent catalytic activity towards benzoylacetonitrile
was identified from a nitrilase tool-box, which was developed previously in our laboratory for (R)-o-chloromandelic acid
synthesis from o-chloromandelonitrile. The HpN was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity
by nickel column affinity chromatography, and its biochemical properties were studied. The HpN was very stable at 30–40 °C,
and highly active over a wide range of pH values (pH 6.0–10.0). In addition, the HpN could tolerate against several hydrophilic
organic solvents. Steady-state kinetics indicated that HpN was highly active towards benzoylacetonitrile, giving a K of 4.2 mM
M
−1
and a kcat of 170 s , the latter of which is ca. fivefold higher than the highest record reported so far. A cascade reaction for the
synthesis of optically pure (S)-β-phenylalanine from benzoylacetonitrile was developed by coupling HpN with an ω-
transaminase from Polaromonas sp. JS666 in toluene-water biphasic reaction system using β-alanine as an amino donor.
Various (S)-β-amino acids could be produced from benzoylacetonitrile derivatives with moderate to high conversions (73–
9
9%) and excellent enantioselectivity (> 99% ee). These results are significantly advantageous over previous studies, indicating
a great potential of this cascade reaction for the practical synthesis of (S)-β-phenylalanine in the future.
.
.
.
.
Keywords Biocatalysis Nitrilase ω-Transaminase Cascade reaction (S)-β-Phenylalanine
Introduction
expensive catalysts or chiral auxiliaries, and careful control of
the reaction conditions. All of the abovementioned drawbacks
limit the practical application of chemical methods.
Optically pure β-amino acids occur in numerous natural prod-
ucts with potent biological activities and represent an impor-
tant class of chiral building blocks for a variety of pharmaceu-
ticals, agrochemicals, and other fine chemicals (Jin et al. 2006;
Ruf et al. 2012). They can also be incorporated into proteins
and peptide mimics to improve their stability and therapeutic
properties (Steer et al. 2002). Due to the significance of
enantiomerically pure β-amino acids, great efforts have been
made towards their efficient synthesis (Weiner et al. 2010).
However, chemical methods are usually performed in kinetic
resolution mode whose theoretical yield is only 50% and typ-
ically require the acquisition of chiral precursors, high loads of
As an alternative, biocatalytic methods are attracting tre-
mendous attention because of the environmentally benign re-
action conditions and high selectivities (chemoselectivity, re-
gioselectivity, and enantioselectivity) (Bornscheuer et al.
2012; Xu et al. 2017). Therefore, several enzymatic routes
for the synthesis of optically pure β-amino acids have been
developed, including kinetic resolution of racemic β-amino
acids or their ester/amide derivatives (Bea et al. 2011; Li
et al. 2013; Weise et al. 2017; Wu et al. 2010; You et al.
2013) and asymmetric amination/transamination of respective
keto acids or asymmetric addition of ammonia to arylacrylates
(Kim et al. 2007; Weise et al. 2015; Zhang et al. 2015a). In the
case of kinetic resolution, the theoretical yield of only 50%
limits their practical application, while in the case of asym-
metric synthesis, the biocatalysts involved suffer from either
low activity (Kim et al. 2007; Weise et al. 2015; Zhang et al.
*
Jian-He Xu
jianhexu@ecust.edu.cn
1
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key
Laboratory of Bioreactor Engineering and Shanghai Collaborative
Innovation Center for Biomanufacturing, East China University of
Science and Technology, 130 Meilong Road, Shanghai 200237,
People’s Republic of China
2015a) or poor regioselectivity/enantioselectivity (Weise et al.
2015), resulting in low yields of the target product.
Additionally, the preparation of racemic β-amino acids and

Appl Microbiol Biotechnol
their derivatives are relatively complicated and highly expen-
sive, which would significantly increase the cost of the pro-
cess. Therefore, there is still great need for the development of
new methods for the asymmetric synthesis of optically pure β-
amino acids from cheap and easily available starting materials.
Nitrilase, which catalyzes the hydrolysis of nitriles to car-
boxylic acids, is considered as one of the most promising
industrial-relevant biocatalysts and has been employed for the
production of optically pure (R)-(–)-mandelic acid at BASF in
an industrial scale (Ress-Loschke et al. 2005). Nitrilases from
different origins have also been applied for the synthesis of
various bulk chemicals and fine chemicals, such as acrylic acid,
lactams, glycolic acid, nicotinic acid, (R)-2-chloromandelic ac-
id, (R)/(S)-4-cyano-3-hydroxybutyric acid, S-ibuprofen, (3S)-3-
cyano-5-methyl hexanoic acid, (R)-ethyl-3-hydroxyglutarate,
as well as (R)/(S)-β-hydroxy acids (Ankati et al. 2009;
DeSantis et al. 2003; Dong et al. 2010; Gavagan et al. 1998;
Nagasawa et al. 1990; Stepanek et al. 1999; Wu et al. 2008; Xie
et al. 2006; Yamamoto et al. 1990; Zhang et al. 2012). The
synthetic applications and future perspectives of nitrilases were
reviewed elsewhere (Gong et al. 2017; Martinkova and Kren
expensive NADPH (Zhang et al. 2015a). Recently, a similar
process coupling a nitrilase and an ω-transaminase for the syn-
thesis of β-amino acids from β-nitriles was reported (Mathew
et al. 2017). The weakness of this process is that the expensive
amino donor, optically pure (S)-phenethylamine, was needed to
achieve a moderate conversion. In addition, the byproduct
acetophenone served as a strong inhibitor for ω-transaminase,
which would reduce the total yield of the product (Bea et al.
2011; Kim et al. 2007).
Materials and methods
Materials
Benzoylacetonitrile (98%) was obtained from Energy
Chemicals (Shanghai, China), racemic β-phenylalanine
(99%) was purchased from Accela ChemBio Co., Ltd.
(Shanghai, China), β-alanine (98%), pyridoxal 5′-phosphate
(PLP, 98%), and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl
isothiocyanate (GITC, 98%) were provided by Aladdin
(Shanghai, China). Cation resin D001 was kindly donated
by Shanghai Huazhen Sci. & Tech. Co., Ltd. All other
chemicals with the highest purity were also obtained commer-
cially and used without further purification unless otherwise
stated.
2010; Martinkova and Mylerova 2003). Recently, we also re-
ported the application of nitrilases from Alcaligenes sp.
ECU0401 and Labrenzia aggregata for the efficient synthesis
of (R)-(–)-mandelic acid and (R)-o-chloromandelic acid, re-
spectively (Zhang et al. 2010, 2012, 2015b).
In the present study, a nitrilase from Hoeflea phototrophica
DFL-43, which shows very high activity towards
benzoylacetonitrile, was identified by screening the nitrilase
tool-box previously developed in our laboratory for (R)-o-
chloromandelic acid production from o-chloromandelonitrile
The cloning and overexpression of nitrilases from different
origins were based on our previous studies (Zhang et al.
2012). Gene sequence of the ω-transaminase from
Polaromonas sp. JS666 (GenBank accession number:
ABE43415.1) as described previously (Bea et al. 2011) was
synthesized by Genscript (Nanjing, China) and cloned into the
vector pET28a (+). The ω-transaminase was overexpressed in
E. coli BL21 (DE3) as described previously (Bea et al. 2011).
(
Zhang et al. 2012), and its biochemical properties were char-
acterized in detail. In addition, a new strategy for the asymmet-
ric synthesis of optically pure β-phenylalanine from
benzoylacetonitrile was developed by coupling the nitrilase-
catalyzed benzoylacetonitrile hydrolysis with an ω-
transaminase-mediated transamination of benzoylacetic acid
Purification of recombinant nitrilase HpN
(
Scheme 1). Although β-amino acids could also be produced
Recombinant E. coli cells expressing HpN were harvested by
centrifugation (8000×g) at 4 °C for 10 min, washed twice by
physiological saline, and resuspended in buffer A (20 mM
sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH
from β-nitriles by a one-pot two-step process employing a
nitrilase and an amino acid dehydrogenase, however, a third
enzyme (glucose dehydrogenase) is needed to regenerate the
Scheme 1 Two-step one-pot
synthesis of (S)-β-phenylalanine
from benzoylacetonitrile

Appl Microbiol Biotechnol
8
.0), then the cells were disrupted by sonication with an ultra-
after being incubated at different temperatures (30, 40, 50, and
60 °C) for a required period of time.
sonic oscillator (JY92-II, Scientz Biotech. Co., Ltd.). The cell
debris was removed by centrifugation (12,000×g) at 4 °C for
4
0 min. Then the supernatant was loaded onto a His-trap Ni-
Influence of metal ions and EDTA on nitrilase activity
NTA FF column (5 mL, GE Healthcare Corp.) pre-
equilibrated with buffer A, and the proteins were eluted with
an increasing gradient of imidazole from 10 to 500 mM in
buffer A at a flow rate of 5 mL/min. The fractions containing
the target protein were concentrated to about 1 mL and ex-
changed with potassium phosphate buffer (20 mM, pH 8.0;
The influences of different metal ions or EDTA on nitrilase
activity were studied by assaying the enzyme activity in the
presence of specific metal ion or EDTA at final concentrations
of 1 and 10 mM. The activity measured in the absence of
metal ions and EDTA was taken as 100% and used as a
reference.
4
× 10 mL) for reducing the imidazole concentration using
centrifugal filter units with 10 kDa cutoff (Millipore, USA).
Protein purity was confirmed by SDS-PAGE analysis, and
protein concentration was determined by the Bradford method
using bovine serum albumin as the standard (Bradford 1976).
Effect of organic solvents on nitrilase activity
The effects of various organic solvents on nitrilase activity
were investigated by measuring the enzyme activity in the
presence of specific organic solvent at a final concentration
of 5% (v/v). The activity measured in the absence of organic
solvent was taken as 100% and used as a reference.
Activity assay
For nitrilase: the reaction mixture (1 mL) containing 10 mM
benzoylacetonitrile (200 mM stock solution in DMSO), 10 μg
of enzyme in potassium phosphate buffer (100 mM, pH 8.0),
was incubated at 30 °C and 1000 rpm on a Thermomixer
Compact (Eppendorf, Germany). Samples (100 μL) were
withdrawn at 10 min, and the reaction was stopped by adding
Substrate spectrum
For substrate specificity investigation, the nitrilase activity
was assayed under the standard assay conditions with
10 mM each substrate (from 200 mM stock solution in
DMSO). The conversion was determined by measuring the
ammonia formed in the reaction as described previously
(Fawcett and Scott 1960). A control experiment, in which
the nitrilase was replaced by reaction buffer, was performed
for each substrate, and the ammonia produced in the control
experiment was subtracted from the value found in the en-
zyme reaction.
1
0 μL HCl (2 M) and extracted with 500 μL ethyl acetate for
GC analysis. The conversion was determined by measuring
the amount of benzoylacetonitrile consumed in the reaction.
One unit of enzyme activity was defined as the amount of
enzyme consuming 1 μmol of benzoylacetonitrile per minute
under the standard assay conditions.
For ω-TA: the reaction mixture (10 mL) containing 20 mM
benzoylacetonitrile in 5 mL toluene, 0.1 mM PLP, 200 mM β-
alanine, 50 mg lyophilized nitrilase powder, and 150 mg of
lyophilized transaminase powder in 5 mL potassium phos-
phate buffer (100 mM, pH 8.0) was incubated at 37 °C and
Kinetic constants
1
3
80 rpm. The aqueous layer (100 μL) was withdrawn at
0 min for HPLC analysis. The conversion was determined
To determine the steady-state kinetic constants of the nitrilase
towards benzoylacetonitrile, the reactions (1 mL) were carried
out at 50 °C and 1000 rpm with substrate concentrations rang-
ing from 1 to 20 mM using purified HpN in potassium phos-
phate buffer (100 mM, pH 8.5). Aliquots (100 μL) were taken
at fixed time intervals, and the reactions were stopped by
adding 10 μL HCl (2 M) and extracted with 500 μL ethyl
acetate for GC analysis. The best fit of the data to the
Michaelis-Menten equation found using the solver function
of Microsoft excel gave the respective kinetic constants.
by measuring the amount of β-phenylalanine produced in the
reaction. One unit of enzyme activity was defined as the
amount of enzyme producing 1 μmol of β-phenylalanine
per minute under the coupled reaction conditions.
Effect of temperature and pH on the activity of HpN
and stability
The effects of temperature and pH on HpN were determined
by measuring the enzyme activity at different temperatures
Two-step one-pot synthesis of (S)-β-phenylalanine
(
1
20–60 °C) and in buffers with different pH values (pH 5.0–
0.0): 100 mM sodium citrate buffer, pH 5.0–6.0; 100 mM
from benzoylacetonitrile
potassium phosphate buffer, pH 6.0–8.0; 100 mM Tris-HCl
buffer, pH 8.0–9.0; and 100 mM glycine-NaOH, pH 9.0–10.0.
Thermostability of the nitrilase was investigated by mea-
suring the residual activity under the standard assay conditions
Asymmetric synthesis of (S)-β-phenylalanine from
benzoylacetonitrile was carried out by a cascade reaction cou-
pling the nitrilase with an ω-transaminase. The reaction mix-
ture (10 mL) containing 200 mM β-alanine, 1 mM PLP,

Appl Microbiol Biotechnol
5
0 mg lyophilized nitrilase powder (0.6 U, determined in the
Enzyme purification
biphasic system), and 150 mg lyophilized ω-transaminase
powder (0.75 U, based on the coupled reaction) in 5 mL po-
tassium phosphate buffer (100 mM, pH 8.0), and 20 mM
benzoylacetonitrile in 5 mL toluene was incubated at 37 °C
and 180 rpm. At different time intervals, samples were with-
drawn for HPLC and GC analysis. For preparative synthesis,
the reaction was performed in 100-mL scale under the same
reaction conditions described above. The product was isolated
by cation exchange resin column as described previously
using D001 instead (Wu et al. 2010).
Due to the N-terminal His-tag, HpN was easily purified to
homogeneity by nickel column affinity chromatography
(Fig. 1), and the purified enzyme migrated as a single band
on SDS-PAGE with a molecular size of about 40 kDa,
which is consistent with that predicted from the amino acid
sequence. The purified nitrilase showed a very high specific
activity of 35 U mg protein towards benzoylacetonitrile,
indicating that the enzyme was purified by 15-fold from the
cell-free extract.
−1
Analytic methods
Effects of pH and temperature
Quantitative analysis of β-phenylalanine was analyzed by
HPLC with an ODS-C18 column (5 μm, 4.6 × 200 mm,
Shimadzu, Japan), eluted with methanol:water:trifluoroacetic
acid (55:45:0.1, v/v/v) at a flow rate of 0.8 mL/min and detect-
ed at 254 nm. For enantiomeric excess (ee) determination,
The activity of the purified HpN was investigated at vari-
ous pH values ranging from 5.0 to 10.0. Interestingly, the
nitrilase was active over a wide range of pH values from
6.0 to 10.0, and more than 70% of the maximum activity
was retained between pH 6.0 and 9.0. The maximum ac-
tivity was observed at 100 mM Tris-HCl buffer with a pH
value of 8.5 (Fig. 2a). The optimum temperature of HpN
was 50 °C, lower or higher temperatures would significant-
ly decrease the nitrilase activity, indicating that the nitrilase
was highly temperature-sensitive, especially at higher tem-
peratures (Fig. 2b).
(S)-β-phenylalanine was derivatized by GITC as described
previously (Ito et al. 1992) before HPLC analysis.
Conversion of benzoylacetonitrile and the concentration of
byproduct acetophenone were determined by GC analysis
with Rxi®-5Sil MS Capillary Column (0.25 μm, 0.25 mm ×
3
0 m, RESTEK, USA) using n-dodecane as an internal stan-
dard. The temperature for injector and detector was 280 °C,
and the column temperature was 100 °C for 2 min, rise to
Thermostability of the purified nitrilase was then deter-
mined at temperatures of 30, 40, 50, and 60 °C, respectively,
in potassium phosphate buffer (100 mM, pH 8.0). It should be
noted that the enzyme was very stable at 30 °C, and no activity
loss was observed after 24-h incubation (Fig. 3). The nitrilase
was relatively stable at 40 °C with a half-life of 12 h, while at
1
60 °C at 10 °C/min and hold for 2 min.
Results
50 and 60 °C, the enzyme was more labile, giving half-lives of
Screening of nitrilases for benzoylacetonitrile
hydrolysis
only 3 and 1 h, respectively.
Influences of metal ions and EDTA
To identify nitrilases capable of catalyzing benzoylacetonitrile
hydrolysis, a nitrilase tool-box previously developed in our
laboratory for the synthesis of (R)-o-chloromandelic acid from
o-chloromandelonitrile (Zhang et al. 2012) was subjected to a
primary screening using benzoylacetonitrile as a substrate
The effects of metal ions and EDTA on the activity of HpN
were investigated at concentrations of 1 and 10 mM. As
shown in Table 2, the nitrilase activity was obviously
+
inhibited by the thiol-binding metal ions, such as Ag ,
2
+
2+
+
2+
(
10 mM) under the standard assay conditions. As shown in
Hg , and Cu , and as low as 1 mM Ag or Hg could
result in completely loss of enzyme activity, confirming
that the thiol group is essential for nitrilase activity; similar
results were also observed by previous studies (Zhang et al.
Table 1, a nitrilase from Hoeflea phototrophica DFL-43, ab-
breviated as HpN, showed the highest hydrolytic activity to-
wards benzoylacetonitrile with a relatively high activity of
.35 U mg− cell-free extract. Three other nitrilases (SpwN,
1
2011a, 2012). Meanwhile, Ni and Fe could also serve
as effective inhibitors. The other metal ions and the metal
chelating agent EDTA did not show obvious effect on the
nitrilase activity.
2+
2+
2
LaN, and ShwN) showed moderate to low activities, ranging
−1
from 0.02 to 0.92 U mg cell-free extract. In contrast, three
more nitrilases (ArN, EbN, and JpE) could not accept
benzoylacetonitrile as substrate at all. It is noteworthy that
all these nitrilases could be expressed very well in E. coli in
soluble form. Therefore, HpN was chosen as a promising
nitrilase for benzoylacetonitrile hydrolysis and was used for
the following research.
Effect of organic solvents
Since most of the nitriles are highly hydrophobic, and in the
bioreaction process, organic solvents are usually added to help

Appl Microbiol Biotechnol
Table 1 Screening of nitrilase for benzoylacetonitrile hydrolysis
a
Nitrilase
Microorganism
GenBank accession number
Activity
U/mg crude enzyme)
b
(
AnN
ArN
EbN
HpN
Arsenophonus nasoniae
DSM 15247
Agrobacterium radiobacter
CGMCC 1.2556
Erwinia billingiae
DSM 17872
FN545161.1
N.D.
NC_011987.1
N.D.
NC_014305.1
N.D.
Hoeflea phototrophica DFL-43
DSM 17068
NZ_ABIA02000005.1
2.35 ± 0.05
JpN
LaN
Alcaligenes sp. CGMCC 2009
FJ943638
N.D.
Labrenzia aggregata
DSM 13394
NZ_AAUW01000019.1
0.34 ± 0.03
ShwN
SpwN
Shewanella woodyi
ATCC 51908
Sphingomonas wittichii RW1
DSM 6014
NC_010506.1
NC_009511.1
0.02 ± 0.00
0.92 ± 0.06
N.D., not detected
a
The microorganisms were obtained from DSMZ, ATCC, or CGMCC
b
The activities of crude enzymes were determined with 10 mM benzoylacetonitrile under the standard assay conditions. One unit of enzyme activity was
defined as the amount of enzyme required for the consumption of 1 μmol benzoylacetonitrile under the standard assay conditions
the dispersion of substrate in the reaction mixture. However,
organic solvents especially highly hydrophilic organic sol-
vents have also been proven to be detrimental to biocatalysts
nitrilase, and more than 80% of the activity could be retained.
The only exception was acetone, in which more than 60% of
the nitrilase activity was inhibited, demonstrating that acetone
(
Zhang et al. 2011b). Therefore, the effects of various organic
solvents on the nitrilase activity were examined with a con-
centration of 5% (v/v). As shown in Fig. 4, most of the organic
solvents tested displayed good biocompatibility with the
120
a
1
00
80
60
40
20
0
M
1
2
4
5
6
7
8
9
10
11
pH
9
7 kDa
6 kDa
6
1
00
b
8
6
4
2
0
0
0
0
0
4
3 kDa
1 kDa
3
1
0
20
30
40
50
60
70
o
Temperature ( C)
Fig. 2 The pH and temperature optima of the purified enzyme. (a) pH
optima. Diamond, 100 mM sodium citrate buffer (pH 5.0−6.0); Square,
1
00 mM potassium phosphate buffer (pH 6.0−8.0); Triangle, 100 mM
2
0 kDa
Tris-HCl buffer (pH 8.0−9.0); Circle, 100 mM glycine-NaOH buffer (pH
9
various temperatures (20−60 °C) in 100 mM potassium phosphate buffer
(pH 8.0)
.0−10.0). (b) Temperature optima: The enzyme activity was measured at
Fig. 1 SDS-PAGE analysis of the purified HpN. M, protein markers;
Lane 1, cell-free extract; Lane 2, purified enzyme

Appl Microbiol Biotechnol
1
1
20
00
120
100
80
8
6
4
2
0
0
0
0
0
60
4
2
0
0
0
0
5
10
15
20
25
Incubation time (h)
Fig. 3 Thermal stability of the purified enzyme. The purified enzyme was
incubated in potassium phosphate buffer at different temperatures for a
required period and the residual activity was measured. Triangle, 30 °C;
Diamond, 40 °C; Square, 50 °C; Circle, 60 °C
Organic solvents (5%, v/v)
Fig. 4 Effect of various organic solvents on the activity of nitrilase
might not be a suitable organic solvent for the nitrilase-
catalyzed reaction.
aromatic ring of phenylacetonitrile and benzoylacetonitrile
significantly decreased the nitrilase activity, probably due to
steric hindrance. It should be noted that the HpN displayed
very low activities towards aliphatic nitriles with the exception
of diaminomaleonitrile, which could not be hydrolyzed by
HpN. In addition, the nitrilase HpN does not accept aromatic
nitriles as substrates.
Substrate spectrum
The substrate specificity of the purified nitrilase HpN towards
a series of nitriles including arylacetonitriles, aromatic nitriles,
and aliphatic nitriles were investigated (Table 3). It is not
surprising that the nitrilase showed moderate to high activities
towards arylacetonitriles since the nitrilase tool-box from
which HpN was identified was developed previously in our
laboratory using an arylacetonitrilase gene sequence as a tem-
plate for blasting in NCBI database (Zhang et al. 2012). The
HpN exhibited very high activity towards phenylacetonitrile
and benzoylacetonitrile. However, substitution on the
Steady-state kinetics
The steady-state kinetic constants of the purified nitrilase
towards benzoylacetonitrile were studied with substrate
concentrations ranging from 1 to 20 mM, giving Vmax, kcat,
Table 3 Substrate spectrum of HpN
Substrate
Relative activity
(%)
a
Table 2 Effects of metal
ions and EDTA on the
a
Reagent
Relative activity (%)
nitrilase activity
Benzoylacetonitrile
100 ± 5
43 ± 3
16 ± 1
38 ± 4
10 ± 1
32 ± 3
7 ± 1
1
mM
10 mM
3
4
-Chlorobenzoylacetonitrile
-Chlorobenzoylacetonitrile
Control
100 ± 3
81 ± 2
108 ± 6
51 ± 4
13 ± 2
0 ± 0
100 ± 5
85 ± 4
100 ± 5
41 ± 3
6 ± 1
2+
3-Methoxylbenzoylacetonitrile
4-Methoxylbenzoylacetonitrile
Mandelonitrile
Mg
2+
Ca
2+
Fe
2+
o-Chloromandelonitrile
Phenylacetonitrile
Cu
+
160 ± 12
38 ± 3
N.D.
Ag
0 ± 0
2+
o-Chlorophenylacetonitrile
Benzonitrile
Hg
0 ± 0
0 ± 0
2+
Mn
85 ± 5
80 ± 2
94 ± 3
89 ± 3
34 ± 2
95 ± 4
76 ± 3
80 ± 2
102 ± 5
85 ± 2
17 ± 1
80 ± 3
2+
3-Cyanopyridine
N.D.
Zn
+
2,3-Dicyanopyrazine
Acetonitrile
N.D.
Li
2+
12 ± 2
18 ± 2
N.D.
Pb
2+
Glycolonitrile
Ni
Diaminomaleonitrile
EDTA
a
N.D., not detected
The enzyme activity was assayed under
the standard assay conditions with one of
the metal ions or EDTA. Control enzyme
activity was assayed in the absence of any
tested additive
a
The enzyme activity was measured under the standard assay conditions
with 10 mM substrate. The conversion was determined by measuring the
amount of ammonia produced as described previously (Fawcett and Scott
1960). The activity towards benzoylacetonitrile was taken as 100%

Appl Microbiol Biotechnol
−
1
−1
Table 4 Biocatalytic two-step one-pot synthesis of various β-amino
acids from benzoylacetonitrile derivatives
and K of 256 U mg , 170 s , and 4.2 mM, respectively.
M
Although nitrilases have found applications in the synthe-
sis of various pharmaceuticals, agrochemicals, and fine
chemicals, their activity levels (Vmax) if available are usu-
Entry
Substrate (X =)
Time (h)
Conversion (%)
ee (%)
a
−
1
1
2
3
4
5
H
12
24
24
24
24
> 99
89
> 99
> 99
> 99
> 99
> 99
ally below 50 U mg (Banerjee et al. 2006; Zhang et al.
011a; Zhu et al. 2007), indicating that the nitrilase pre-
b
3-Cl
2
b
4-Cl
77
sented in this study represents a highly active and promis-
ing biocatalyst.
b
3-OMe
86
b
4-OMe
73
a
The biocatalyst loadings were 10 mg/mL lyophilized nitrilase powder
Two-step one-pot synthesis of (S)-β-amino acids
from benzoylacetonitrile derivatives
and 30 mg/mL lyophilized ω-transaminase powder
b
The biocatalyst loadings were 20 mg/mL lyophilized nitrilase powder
and 60 mg/mL lyophilized ω-transaminase powder
Due to the significant inhibitory effect of carbonyl compounds
on ω-transaminase (Bea et al. 2011; Kim et al. 2007) and the
poor solubility of benzoylacetonitrile derivatives in aqueous
buffer, a toluene-aqueous biphasic reaction system was
adopted for the two-step one-pot synthesis of (S)-β-amino
acids from benzoylacetonitrile derivatives since this biphasic
system has been proven very useful in relieving substrate in-
hibition (Zhang et al. 2011a, 2012). Meanwhile, toluene is
also a good solvent for benzoylacetonitrile derivatives. In the
toluene-aqueous biphasic reaction system (1:1, v/v), 20 mM
benzoylacetonitrile in the organic phase could be completely
transformed into (S)-β-phenylalanine within 12 h with an op-
tical purity of > 99% ee (Fig. 5 and Table 4). However, only
moderate conversions (73–89%) of (S)-β-amino acids
were obtained from benzoylacetonitrile derivatives even
though the biocatalyst loading was twofold higher than that
for benzoylacetonitrile, and the reaction time was also ex-
tended to 24 h, which was ascribed to the much lower
specific activity of the nitrilase towards benzoylacetonitrile
derivatives (Table 3). A preparative scale reaction
after purification by column chromatography using a cat-
ion exchange resin (D001).
Discussion
Due to the important role of optically pure β-amino acids in
synthetic organic chemistry and the common limitations in-
cluding poor catalytic activity, unsatisfactory selectivity, and
the low product yield of currently available biocatalytic syn-
thesis methods (Bea et al. 2011; Kim et al. 2007; Li et al. 2013;
Weise et al. 2015, 2017; Wu et al. 2010; You et al. 2013;
Zhang et al. 2015a), there is still a great need to develop
new synthetic routes, especially asymmetric synthetic
methods starting from cheap raw materials. ω-Transaminase
has been shown to be a group of versatile biocatalyst in the
synthesis of chiral amines or amino acids due to its widespread
in nature and broad substrate spectrum (Koszelewski et al.
2010). Some progresses have been made towards the asym-
metric amination of β-ketoacids for the synthesis of optically
pure β-phenylalanine; however, the inherent inhibitory effect
of β-ketoacids on ω-transaminase and the spontaneous decar-
boxylation to acetophenone and carbon dioxide would greatly
reduce the product yield (Bea et al. 2011; Kim et al. 2007).
Although this could be partially alleviated by coupling the
transamination with lipase, in which the more stable β-
ketoacid esters were used instead of the labile β-ketoacids
(
100-mL) was then carried out under the same reaction
conditions, and enantiomerically pure (S)-β-phenylalanine
was obtained with an isolated yield of 82 and > 99% ee
2
1
1
1
2
8
4
0
6
2
2
1
8
6
4
2
0
00
0
0
(Bea et al. 2011; Kim et al. 2007), the preparation of the ester
0
precursors is relatively complicated and costly, since either
harsh reaction conditions (e.g., − 78 °C) (Galliford and
Scheidt 2008) or highly expensive catalysts (e.g., palladium)
(Chen et al. 2013) were required.
To this end, we focused on the cascade reaction coupling
nitrilase with ω-transaminase, in which the cheap and easily
available benzoylacetonitrile was hydrolyzed by nitrilase into
benzoylacetic acid which was then aminated by ω-
transaminase forming β-phenylalanine (Scheme 1).
Although nitrilases have been employed for the synthesis of
0
-
0
2
4
6
8
10
12
Reaction time (h)
Fig. 5 Time course for the two-step one-pot synthesis of (S)-β-phenylal-
anine from benzoylacetonitrile in a biphasic system. Triangle, concentra-
tion of (S)-β-phenylalanine; Diamond, concentration of
benzoylacetonitrile; Square, concentration of acetophenone; Circle, the
ee of (S)-β-phenylalanine

Appl Microbiol Biotechnol
various carboxylic acids from nitriles, there was only one
nitrilase (NitBJ) reported to be active towards
benzoylacetonitrile so far. Most importantly, the specific ac-
added to the reaction mixture to help the distribution of
substrates.
A cascade reaction coupling nitrilase with ω-transaminase
for the enzymatic synthesis of optically pure β-phenylalanine
from benzoylacetonitrile was thereby firstly performed in an
aqueous monophasic system. However, only little amount of
β-phenylalanine was detected, while benzoylacetic acid and
acetophenone accounted the majority of products (data not
shown), indicating that benzoylacetonitrile was successfully
hydrolyzed by nitrilase to form benzoylacetic acid, and
acetophenone was formed by the spontaneous decarboxyl-
ation of benzoylacetic acid. The low yield of β-
phenylalanine might be ascribed to the inhibitory effect of
carbonyl compounds on ω-transaminase, which was also ob-
served in previous studies (Bea et al. 2011; Kim et al. 2007).
In addition, when benzoylacetonitrile or acetophenone was
used as the substrate for the ω-transaminase-catalyzed trans-
amination, neither decrease of substrate nor formation of new
product was detected, demonstrating that these two com-
pounds could not be accepted as substrates by the ω-
transaminase.
To relieve the substrate inhibition, a toluene-water biphasic
system (1:1, v/v) was then adopted for the cascade reaction.
Meanwhile, excess amount of ω-transaminase as compared
with nitrilase was added to make sure that benzoylacetic acid
produced at the first step could be transformed into β-
phenylalanine by ω-transaminase as soon as its formation
and the spontaneous decarboxylation to acetophenone could
be avoided. Under these reaction conditions, various optically
pure (S)-β-amino acids could be produced from
benzoylacetonitrile derivatives in the cascade reaction
employing nitrilase and ω-transaminase with moderate to
high yields (Table 4). Although similar cascade reactions cou-
pling lipase/nitrilase with ω-transaminase have also been
employed for the production of a series of (S)-β-amino acids,
only low to moderate yield of the product could be obtained
tivity of NitBJ towards benzoylacetonitrile was merely
1
0
2
.36 U mg− protein (Mathew et al. 2017; Zhang et al.
015a). By screening the nitrilase tool-box developed in our
laboratory (Zhang et al. 2012), a Hoeflea phototrophica
nitrilase (HpN) was identified, exhibiting excellent catalytic
activity towards benzoylacetonitrile with a V
2
of
max
56 U mg− protein, which is about fivefold higher than the
1
highest nitrilase activity reported so far (Zhu et al. 2007).
Substrate spectrum investigation showed that HpN was highly
active towards arylacetonitriles, while very low activities were
observed with aliphatic nitriles, and aromatic nitriles could not
be hydrolyzed by HpN. A BLASTp search revealed that the
amino acid sequence of HpN shows 67% identity to a nitrilase
from an uncultured organism (GenBank accession number:
AAR97377.1) (Robertson et al. 2004), which is the highest
among the homologous nitrilases, indicating that HpN might
represent a novel nitrilase. The nitrilase HpN was then chosen
for the following research due to its highest activity and good
expression level in E. coli.
Biochemical property characterization indicated that the
nitrilase was very stable between 30 and 40 °C (Fig. 3), which
are frequently employed temperatures for the biotransforma-
tion process. It should be noted that the nitrilase was highly
active over a wide pH range (6.0–10.0), with an optimum pH
around 8.5 (Fig. 2a). The optimum temperature (37 °C) and
pH (8.5) of ω-transaminase from Polaromonas sp. JS666
(
Bea et al. 2011) are similar to those of HpN, which is bene-
ficial for the cascade reaction since coordination of the inter-
compatibility between different enzymes in the cascade pro-
cess is a great challenge. In addition, the nitrilase could toler-
ate against a wide range of hydrophilic organic solvents (Fig.
4), which is very important for the biotransformation of highly
hydrophobic nitriles, in which organic solvents are usually
Table 5 Comparison of different
cascades for the synthesis of (S)-
β-phenylalanine
Substrates
Substrate loading
mM)
Reaction time
(h)
Conversion
(%)
Ref.
(
a
Ethyl 3-oxo-3-phenyl
propionate
10
24
24
24
24
18
24
12
20
Kim et al. (2007)
Bea et al. (2011)
Bea et al. (2011)
Mathew et al. (2016a)
Mathew et al. (2016b)
Mathew et al. (2017)
This study
b
10
10
50
20
18
c
52
d
96
d
43.6
d
Benzoylacetonitrile
10
72
c
2
0
> 99
a
b
c
d
3
-Aminobutyric acid was used as the amino donor
L-Alanine was used as the amino donor
β-Alanine was used as the amino donor
(S)-phenethylamine was used as the amino donor

Appl Microbiol Biotechnol
Banerjee A, Kaul P, Banerjee UC (2006) Purification and characterization
of an enantioselective arylacetonitrilase from Pseudomonas putida.
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(
Table 5), probably due to the much lower catalytic activity of
the nitrilase towards the nitrile compounds than that used in
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2
016a, b, 2017). Another possible reason might be the
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of the keto nitriles and regulating the substrate concentration
around the biocatalyst and minimizing the substrate inhibition.
The only limitation of the aqueous-toluene biphasic system
might be the mass transfer problem (partially due to high
biocatalyst loading in this study); therefore, selection of suit-
able organic solvents for the biphasic system or using extrac-
tion reagents is still needed in the future studies. It should also
be noted that the much cheaper amino donor β-alanine was
used in this study instead of the highly expensive (S)-
phenethylamine employed in the previous studies (Bea et al.
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2
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would significantly reduce the cost of the bioprocess.
In summary, a novel nitrilase from Hoeflea phototrophica
was identified from a home-made nitrilase tool-box, with an
excellent activity towards benzoylacetonitrile which enables
the development of a cascade reaction coupling nitrilase with
ω-transaminase for the enzymatic synthesis of optically pure
(
S)-β-phenylalanine from the cheap and widely available
benzoylacetonitrile. It should be noted that an in-depth opti-
mization of the reaction conditions or protein engineering of
the involved nitrilase and especially ω-transaminase to im-
prove their catalytic efficiencies is still needed to further en-
hance the productivity of the cascade enzymatic process.
However, the much better results in this work than the previ-
ous studies indicate that the cascade reaction we developed in
this study might serve as a promising alternative for the green
production of optically pure (S)-β-phenylalanine in the future.
6
9–81
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enantiomers of thiol compounds by reversed-phase liquid chroma-
tography using chiral derivatization with 2,3,4,6-tetra-O-acetyl-β-d-
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Funding This study was funded by National Natural Science Foundation
of China (nos. 21406067 and 21536004), Natural Science Foundation of
Shanghai (no. 18ZR1408400), Fundamental Research Funds for the
Central Universities (no. 22221818014) and Shanghai Commission of
Science and Technology (no. 15JC1400403).
1
0660–10661
Kim J, Kyung D, Yun H, Cho BK, Seo JH, Cha M, Kim BG (2007)
Cloning and characterization of a novel β-transaminase from
Mesorhizobium sp. strain LUK: a new biocatalyst for the synthesis
of enantiomerically pure β-amino acids. Appl Environ Microbiol
7
3:1772–1782
Koszelewski D, Tauber K, Faber K, Kroutil W (2010) ω-Transaminases
for the synthesis of non-racemic α-chiral primary amines. Trends
Biotechnol 28:324–332
Li D, Ji L, Wang X, Wei D (2013) Enantioselective acylation of β-
phenylalanine acid and its derivatives catalyzed by penicillin G
acylase from Alcaligenes faecalis. Prep Biochem Biotechnol 43:
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
2
07–216
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Mathew S, Jeong SS, Chung T, Lee SH, Yun H (2016a) Asymmetric
synthesis of aromatic β-amino acids using ω-transaminase:
Optimizing the lipase concentration to obtain thermodynamically
unstable β-keto acids. Biotechnol J 11:185–190
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