Development of nitrilase-mediated process for phenylacetic acid production from phenylacetonitrile

Article abstract of DOI:10.1007/s11696-017-0192-x

This study aimed at developing an efficient biotransformation process for phenylacetic acid production from phenylacetonitrile by using recombinant Escherichia coli M15 harboring a double mutant MG nitrilase (I113M/Y199G) from Burkholderia cenocepacia J2315. A yield of 2310 U/mL nitrilase was obtained by fermentation after the optimization of cultivation conditions, with a specific activity of 64 U/mg dcw. The MG nitrilase showed high substrate tolerance and completely hydrolyzed 100 mM phenylacetonitrile in 30 min under optimal conditions. To alleviate substrate inhibition, periodic or continuous batch-feeding of substrate was used during the biotransformation. Up to 164 g/L substrate was completely hydrolyzed in 9 h with continuous batch-feeding using resting cells, corresponding to 400 U/mL of nitrilase activity, and leading to production of 163.4 g/L phenylacetic acid. The hydrolysis process has potential application for phenylacetic acid production on a large scale.

Full text of DOI:10.1007/s11696-017-0192-x

Chem. Pap.
DOI 10.1007/s11696-017-0192-x
ORIGINAL PAPER
Development of nitrilase-mediated process for phenylacetic acid
production from phenylacetonitrile
Haiyang Fan1 Lifeng Chen1 Huihui Sun2 Hualei Wang1 Qinghai Liu1
•
•
•
•
•
Yuhong Ren1 Dongzhi Wei1
•
Received: 25 February 2017 / Accepted: 5 May 2017
Ó Institute of Chemistry, Slovak Academy of Sciences 2017
Abstract This study aimed at developing an efficient
biotransformation process for phenylacetic acid production
from phenylacetonitrile by using recombinant Escherichia
coli M15 harboring a double mutant MG nitrilase (I113M/
Y199G) from Burkholderia cenocepacia J2315. A yield of
2310 U/mL nitrilase was obtained by fermentation after the
optimization of cultivation conditions, with a specific
activity of 64 U/mg dcw. The MG nitrilase showed high
substrate tolerance and completely hydrolyzed 100 mM
phenylacetonitrile in 30 min under optimal conditions. To
alleviate substrate inhibition, periodic or continuous batch-
feeding of substrate was used during the biotransformation.
Up to 164 g/L substrate was completely hydrolyzed in 9 h
with continuous batch-feeding using resting cells, corre-
sponding to 400 U/mL of nitrilase activity, and leading to
production of 163.4 g/L phenylacetic acid. The hydrolysis
process has potential application for phenylacetic acid
production on a large scale.
Introduction
Phenylacetic acid is an important intermediate with a high
demand in the pharmaceutical and perfume industries, and
the major application of phenylacetic acid is as a key
precursor for the synthesis of penicillin G (Zhu et al. 2011).
Several methods have been developed to produce pheny-
lacetic acid. The classic industrial process is the pheny-
lacetonitrile hydrolysis method, which requires the
presence of mineral acids at temperatures of 80–250 °C
(Kagawa et al. 1980).
Alternative biotechnology methods for phenylacetic
acid production have been reported. Koma et al. (2012)
reported a metabolically engineered Escherichia coli strain
with an expanded shikimate pathway for production of
phenylacetic acid. Another potential route for phenylacetic
acid production was by a fermentation process mediated by
Pseudomonas or Comamonas using phenylalanine as
starting material (Farbood et al. 1995). Bertokova et al.
(2015) and Mihal et al. (2016, 2017) used whole-cell
Gluconobacter oxydans as a biocatalyst for the production
of phenylacetic acid through 2-phenylethanol oxidation.
However, these methods had limited applications in
industry due to low production efficiency.
Keywords Biotransformation Á Fed-batch reaction Á
Fermentation Á Nitrilase Á Phenylacetic acid
Nitriles constitute a versatile class of intermediates for
the production of various pharmaceuticals and fine chem-
icals (Banerjee et al. 2002; de Oliveira et al. 2013; Kiel-
basinski et al. 2008). Compared to chemical transformation
methods, nitrilases allow nitrile hydrolysis to occur under
mild conditions of temperature and pH (Vergne-Vaxelaire
et al. 2013; Xue et al. 2016; Zhu et al. 2008). In recent
years, nitrile hydrolysis by nitrilase has been successfully
applied for the commercial production of carboxylic acids
(Wang 2015). Therefore, the biotransformation of
& Hualei Wang
hlwang@ecust.edu.cn
1
State Key Laboratory of Bioreactor Engineering, New World
Institute of Biotechnology, East China University of Science
and Technology, Shanghai 200237, People’s Republic of
China
2
Yellow Sea Fisheries Research Institute, Chinese Academy
of Fishery Sciences, Qingdao 266071, Shandong, People’s
Republic of China
123

Chem. Pap.
phenylacetonitrile by nitrilase could be a promising method
for production of phenylacetic acid.
tone and 30 g/L yeast extract) was used for continuous
feeding of carbon and nitrogen sources during the fer-
mentation period. The feeding started with an initial speed
of 10 mL/h when the optical density at 600 nm (OD600) of
the culture reached 4–4.5. Ammonia was used to maintain
the pH above 7.0. The growing cells were induced with
0.1 mM isopropyl-b-D-thiogalactoside (IPTG) when the
OD600 reached 20. The cultivation temperature was
reduced to 30 °C immediately before the induction. After
induction, the supplementary medium feeding rate was
In our previous work,
a novel nitrilase from
Burkholderia cenocepacia J2315 (BCJ2315) was discov-
ered, and a double mutant MG (I113M/Y199G) that had
higher activity for the hydrolysis of arylacetonitriles was
identified (Wang et al. 2013, 2015). In the present work, for
biotransformation of phenylacetonitrile to phenylacetic
acid using resting cells of E. coli expressing MG as a
biocatalyst, optimal reaction conditions were determined,
including the co-solvents, concentrations of substrate and
resting cells, pH, and temperature. Then, to alleviate sub-
strate inhibition, a fed-batch reaction system with a peri-
odic or continuous substrate feeding in a mono aqueous
phase system was developed.
increased to 20 mL/h. The dry cell weight (dcw), OD600
,
and nitrilase activity were determined by taking samples at
different time points.
Nitrilase activity assay
The reaction mixture (5 mL) containing sodium phosphate
buffer (100 mM, pH 8.0), ethanol (10% v/v), and an
appropriate amount of resting cells was incubated at 30 °C
for 10 min. The reaction was then initiated by adding
100 mM phenylacetonitrile. Samples were periodically
withdrawn and quenched by the addition of 10% (v/v)
diluted HCl (2 M). The concentration of phenylacetic acid
was determined by high-performance liquid chromatogra-
phy (HPLC). One unit of nitrilase activity is defined as the
amount of nitrilase producing 1 lmol phenylacetic acid per
minute under the assay conditions.
Experimental
Materials
Phenylacetonitrile and phenylacetic acid were purchased
from Aladdin Co., Ltd. (Shanghai, China). Tryptone and
yeast extract powder were purchased from Oxoid Ltd.
(Cambridge, UK). All the other chemicals used in this
study were of analytical grade.
Optimization of culture media and conditions
Effect of reaction temperature and pH
A medium named FM designed based on Terrific Broth
(TB) was used for cultivation of the recombinant E. coli/
MG cells. First, the cultivation temperature after induction
and the IPTG concentration were optimized. Then FM
medium (12 g/L tryptone, 16 g/L yeast extract, 24.75 g/L
K2HPO4, 3.47 g/L KH2PO4, 7 mM glycerol, 10 mM
MgCl2) was used as the fermentation medium for high-
density cultivation of the E. coli/MG in a bioreactor. Dif-
ferent glycerol concentrations in FM medium were used for
optimization. Ampicillin (0.1 mM) and kanamycin
(0.05 mM) were added to all media to maintain selection
pressure during cultivation of E. coli/MG.
The determination of optimal reaction temperature and pH
was carried out by incubating the E. coli/MG with
phenylacetonitrile (100 mM) at different temperatures
(20–50 °C) or in buffers with different pH values (100 mM
sodium phosphate buffer for pH 6.0–8.0 and 100 mM Tris–
HCl buffer for pH 8.5–8.9). The thermostability was
investigated by incubation of the E. coli/MG at different
temperatures (20–50 °C) in 100 mM sodium phosphate
buffer (pH 8.0).
Effect of co-solvent
Cultivation of the recombinant E. coli M15/MG
in a fermenter
The reaction mixture (5 mL) containing sodium phosphate
buffer (100 mM, pH 8.0), resting cells (100 mg), and the
organic solvent (10% v/v) was incubated at 30 °C for
10 min and then the reaction was initiated by addition of
phenylacetonitrile to 100 mM final concentration. The
reaction was terminated after 10 min and the conversion
extent was determined. In some cases, ethanol (5–25% v/v)
was added as co-solvent for investigation of the effects of
ethanol concentration on the conversion.
Cultivation was performed in a volume of 2 L in a 7-L
fermenter. A 100-mL seed culture was incubated in LB
broth in a shake flask at 37 °C, 200 rpm for 8 h and then
inoculated into the fermenter. The initial parameters were
set at pH 7.2, 37 °C, a stir rate of 300 rpm, and an aeration
rate of 0.5 vvm. A supplementary medium (40 g/L tryp-
123

Chem. Pap.
Effect of phenylacetonitrile and resting cell
concentrations on phenylacetic acid production
confirmed that most MG nitrilase was expressed as inclu-
sion bodies (data was not shown). To enhance recovery of
the soluble form of MG nitrilase, cultivation of the E. coli/
MG was performed at lower temperatures. Compared to
cultivation at 20 °C, the maximum cell mass and nitrilase
production were both obtained at 30 °C. Therefore, 30 °C
was chosen as the optimal cultivation temperature.
The hydrolysis was performed with substrate concentra-
tions ranging from 25 to 150 mM in a 5-mL reaction
system which contained sodium phosphate buffer
(100 mM, pH 8.0), the resting cells (400 U/mL), and
ethanol (10% v/v) at 30 °C for 10 min. The concentration
of product was determined by HPLC analysis. To obtain
complete conversion, different concentrations of resting
cells (200–600 U/mL) were used in the reaction and the
conversion was determined after 30 min of hydrolysis.
Effect of IPTG concentration in induction
To determine the optimal concentration of IPTG for
induction during the cultivation period, varying concen-
trations of IPTG (0.025, 0.05, 0.1, 0.2, and 0.4 mM) were
used for induction (Table 2). First, in the IPTG concen-
tration range of 0.025–0.1 mM, nitrilase production
increased with increasing IPTG concentration, indicating
that the IPTG concentration for culture induction was not
saturated. In the IPTG concentration range of 0.1–0.4 mM,
a reduction of nitrilase production was observed, which
was probably caused by IPTG inhibition of cell growth.
Maximum nitrilase production and specific activity were
obtained at 0.1 mM IPTG. Therefore, 0.1 mM IPTG was
used as the optimal induction concentration.
Fed-batch production of phenylacetic acid
The fed-batch reaction (300-mL scale) with periodic or
continuous substrate feeding mode was performed in a
500-mL three-necked round-bottom flask. The reaction
temperature was maintained at 30 °C in a water bath. A
pH auto-controller was used to maintain the pH value
between 7.9 and 8.1 by addition of ammonia. The agita-
tion speed was set at 300 rpm. The reaction mixture
(300 mL) containing sodium phosphate buffer (100 mM,
pH 8.0), resting cells (400 U/mL), and ethanol (10% v/v)
was incubated at 30 °C for 20 min; then the reaction was
initialized by addition of 100 mM substrate. In periodic
substrate feeding mode, phenylacetonitrile (100 mM) was
fed into the reaction mixture when the last batch of
substrate was completely converted. In continuous sub-
strate feeding mode, phenylacetonitrile feeding was star-
ted after 30 min with a rate of 23 mL/L h. To monitor the
reaction process, samples were taken at intervals and
analyzed by HPLC.
Optimization of medium composition
In order to obtain a large quantity of the protein of interest,
a modified fermentation medium (FM) was designed based
on TB broth. The effect of glycerol concentration on
nitrilase production was investigated (Table 3). Increasing
the glycerol concentration led to an apparent enhancement
of cell mass. However, excess glycerol (15 mM) resulted in
reduced nitrilase production. Maximum nitrilase produc-
tion was obtained with addition of 7 mM glycerol. Com-
paring addition of 7 mM glycerol with no addition,
although the specific activity was reduced to approximately
86%, more than twofold greater cell mass was obtained.
Taking into account the effects on nitrilase production,
specific activity, and cell mass, addition of 7 mM glycerol
to the FM medium was chosen for high cell density
cultivation.
Analytical methods
The formation of phenylacetic acid was analyzed by HPLC
using a Zorbax SB-Aq column (4.6 mm 9 250 mm, 5 lm)
(Agilent Technologies, Ltd., USA) eluted with methanol/
water/phosphoric acid (40:60:0.1 v/v/v) with a flow rate of
0.8 mL/min. Peaks were detected with a UV detector at
210 nm.
Fermentation in a bioreactor
Results and discussion
Based on the optimization results in shake flasks, 2-L fed-
batch fermentation was performed in a 7-L fermenter to
further enhance nitrilase production and cell mass. The
growth and nitrilase production profiles of E. coli/MG are
shown in Fig. 1. The maximum nitrilase production was
obtained after 28 h of fermentation, and then a decrease of
nitrilase production was observed. Finally, cell mass of
35.9 mg/mL dcw and 2310 U/mL nitrilase production
were obtained. Compared to the results obtained with shake
Effect of temperature in cultivation
To determine the optimal temperature for MG expression,
the effects of cultivating the recombinant cells at 20, 30,
and 37 °C were investigated (Table 1). The minimum
nitrilase production was obtained at 37 °C after 28 h of
cultivation. SDS-PAGE analysis of the resting cells
123

Chem. Pap.
Table 1 Effect of temperature on the growth and nitrilase production of recombinant E. coli
Temperature (°C)
OD600
Cell mass (mg dcw/mL)
Specific activity (U/mg dcw)
Production (U/mL)
20
30
37
4.43
5.12
4.62
5.11
5.39
4.23
77.4
92.4
35.7
395.3
498.0
151.0
E. coli were cultivated in FM medium in a flask at 37 °C before induction by IPTG at the indicated temperature
Table 2 Effect of IPTG concentration on the growth and nitrilase production of recombinant E. coli
IPTG concentration (mM)
OD600
Cell mass (mg dcw/mL)
Specific activity (U/mg dcw)
Production (U/mL)
0.025
0.05
0.1
6.72
6.14
5.12
5.62
5.60
6.41
5.65
5.39
5.45
5.42
60.3
77.7
92.4
69.7
70.0
396.7
438.8
498.0
379.7
379.5
0.2
0.4
E. coli were cultivated in FM medium in a flask for 28 h at 30 °C
Table 3 Effect of medium components on the growth and nitrilase production of recombinant E. coli
Medium
OD600
Cell mass (mg dcw/mL)
Specific activity (U/mg dcw)
Production (U/mL)
FM
5.12
7.04
8.16
9.86
5.39
12.09
13.87
16.69
92.4
79.5
61.2
52.0
498.0
961.2
848.6
867.9
FM ? 7 mM glycerol
FM ? 15 mM glycerol
FM ? 30 mM glycerol
E. coli were cultivated at 30 °C for 28 h. FM medium used here represents the components without glycerol
Effect of reaction temperature and pH
The nitrilase showed relatively high activity with pheny-
lacetonitrile as substrate between 30 and 50 °C, and the
maximum activity was observed at 37 °C (Fig. 2a). Fur-
thermore, the thermostability of the nitrilase was deter-
mined over a period of 12 h (Fig. 2b). Although 37 °C was
the optimal temperature for the MG activity, the activity
was not as stable at 37 °C compared with 30 °C. About 19
and 4% of nitrilase activity were lost during 12 h incuba-
tion at 37 and 30 °C, respectively. Therefore, considering
the importance of the stability of nitrilase activity for the
bioconversion of phenylacetonitrile over an extended per-
iod, 30 °C was chosen as the optimal temperature for the
fed-batch reaction experiments.
Fig. 1 Escherichia coli/MG fermentation in 7-L fermenter. The
symbols represent: squares dcw; and circles nitrilase production.
Maximum dcw and nitrilase production were obtained at 28 h as
35.0 mg/mL and 2310 U/mL, respectively
High activity was maintained over the pH range from
7.0 to 8.5, with the minimum relative activity observed
(89.2%) at pH 8.5 (Fig. 2c). Furthermore, the MG activity
showed good pH stability over the pH range of 7.0–8.5
(data not shown). Therefore, for phenylacetonitrile bio-
conversion, the pH of the reaction mixture was maintained
at approximately 8.0.
flasks, approximate increases of threefold in cell mass and
2.4-fold in nitrilase production were observed with a 7-L
fermenter.
123

Chem. Pap.
bFig. 2 a Effect of temperature on the conversion of phenylacetoni-
trile to phenylacetic acid. The activity at 37 °C was taken to be 100%
(64.3 U/mg). b Thermostability of the nitrilase. Circles 30 °C,
squares 37 °C, triangles 50 °C. The activity at initial time was taken
as 100% (60.2, 64.3, 58.8 U/mg for 30, 37 and 50 °C, respectively).
c Effect of pH on the conversion of phenylacetonitrile to phenylacetic
acid. pH 6.0–8.0, 100 mM sodium phosphate buffer; pH 8.5–8.9,
100 mM Tris–HCl buffer. The activity at pH 8.0 was taken to be
100% (64.3 U/mg)
Effect of co-solvent
In order to enhance the catalytic efficiency, six water-
miscible solvents, methanol, ethanol, isopropanol, ace-
tone, dimethyl sulfoxide (DMSO), and tetrahydrofuran
(THF), were used for the testing the effects of co-sol-
vents on the conversion of phenylacetonitrile by
increasing the solubility of the substrate (Fig. 3a).
Alcohols showed the best compatibility with the nitri-
lase, especially as 100% conversion was achieved in the
presence of methanol and ethanol. Because of environ-
mental and toxicology considerations, ethanol was cho-
sen as the co-solvent for the biotransformation. The
optimal ethanol concentration in the reaction mixture
was determined (Fig. 3b). The phenylacetonitrile could
be hydrolyzed completely at ethanol concentrations up to
15% (v/v). Further increase in the ethanol concentration
present in the reaction led to decreased conversion
efficiency.
Effects of phenylacetonitrile and resting cell
concentration
To determine the tolerance of the resting cells to pheny-
lacetonitrile, the biotransformation reaction was performed
with varying substrate concentrations in an aqueous system
containing 10% (v/v) ethanol. Up to a phenylacetonitrile
concentration of 100 mM, complete hydrolysis was
achieved in 30 min (Fig. 4a). Further increase in the sub-
strate concentration resulted in inhibition of conversion.
Hydrolysis of 100 mM phenylacetonitrile was measured at
nitrilase concentrations of 200–600 U/mL to determine a
suitable catalyst concentration. Complete conversion of the
substrate was achieved with 400 U/mL nitrilase in 30 min
(Fig. 4b). 300 U/mL of nitrilase addition resulted in 97.7%
conversion of the substrate. Therefore, 100 mM substrate
with 400 U/mL of nitrilase was chosen for the fed-batch
reaction experiments.
123

Chem. Pap.
Fig. 3 Effect of co-solvents on
the conversion of
phenylacetonitrile to
phenylacetic acid. a Effect of
different co-solvents. b Effect of
ethanol concentration. The
100% conversion was defined as
100 mM phenylacetonitrile
completely hydrolyzed in
10 min under the described
nitrilase assay conditions
Fig. 4 a Effect of phenylacetonitrile concentration on phenylacetic
acid production. Asterisks 25 mM, squares 50 mM, triangles 75 mM,
circles 100 mM, diamonds 150 mM. b Effect of nitrilase activity
dosage on phenylacetonitrile conversion. The 100% conversion was
defined as 100 mM phenylacetonitrile completely hydrolyzed in
10 min under the described nitrilase assay conditions
Fed-batch production of phenylacetic acid
In the continuous fed-batch mode, the phenylace-
tonitrile feeding rate was set at 200 mM/h to keep the
concentration of substrate in the reaction system below
100 mM, which should alleviate the nitrilase inhibition
observed at high substrate concentrations. A total of
164 g/L phenylacetonitrile was completely hydrolyzed in
9 h, and a measured concentration of 1.2 M (163.4 g/L)
phenylacetic acid was obtained because of the reaction
volume expansion caused by substrate and ammonia
addition (Fig. 5b). During the first 6 h, the nitrilase
activity barely decreased, and a high rate of phenylace-
tonitrile hydrolysis was maintained. After 6 h of sub-
strate feeding, the hydrolysis rate clearly decreased.
Thus, substrate feeding was stopped after 8 h, and an
additional hydrolysis period (1 h) was continued to
guarantee complete hydrolysis.
The catalysis efficiency of the MG nitrilase showed a
reduction if the concentration of the phenylacetonitrile in
the reaction mixture was higher than 100 mM. Hence, a
period fed-batch mode was designed to maintain hydrolysis
performance at a low substrate concentration. In this mode,
100 mM phenylacetonitrile was fed into the reaction mix-
ture when the last substrate was completely converted.
Using this mode, ten batch additions were performed in 8 h
and 117.1 g/L of phenylacetonitrile was hydrolyzed
(Fig. 5a). Reaction volume expansion caused by the sub-
strate and ammonia addition resulted in the production of
0.9 M (122.5 g/L) of phenylacetic acid. During the first
eight batches, the hydrolysis rate barely decreased. Due to
the decrease in nitrilase activity during the reaction period,
the reaction time was extended to ensure complete substrate
hydrolysis for the ninth and tenth batches. Considering the
biotransformation efficiency, the substrate feeding was
terminated after the tenth batch.
At a comparable feeding rate, higher product concen-
tration was obtained by the continuous fed-batch mode
than that obtained by the periodic fed-batch mode. In
addition, the continuous fed-batch reaction mode was more
123

Chem. Pap.
Fig. 5 Time course of
phenylacetonitrile hydrolysis
with fed-batch mode (300-mL
scale). a Periodic substrate
feeding mode. Phenylacetic acid
and phenylacetonitrile
concentrations in the reaction
are denoted by circles and
squares, respectively.
b Continuous substrate feeding
mode
Bertokova A, Vikartovska A, Bucko M, Gemeiner P, Tkac J, Chorvat
D, Stefuca V, Nedela V (2015) Biooxidation of 2-phenylethanol
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efficient than the periodic fed-batch reaction mode (18.2 g/
L h compared to 15.3 g/L h, respectively) and more con-
venient to operate. Using the continuous fed-batch reaction
mode with E. coli/MG, high-volume productivity (163.4 g/
L) of phenylacetic acid was obtained. Compared with the
nitrilase-mediated route, there are several cases for bio-
catalytic production of phenylacetic acid. However, these
reported processes were quite different, such as double
enzymatic cascade reaction process (Mihal et al. 2016),
2-phenylethanol oxidation catalysis by whole-cell G. oxy-
dans (Bertokova et al. 2015; Mihal et al. 2017), and
metabolically process (Koma et al. 2012). Among them,
the highest phenylacetic acid concentration (25 g/L) was
obtained by whole-cell G. oxydans mediated 2-pheny-
lethanol oxidation process in 7 days. Compared to these
mentioned process, the nitrilase-mediated process demon-
strated great potential for industrial production of pheny-
lacetic acid.
Conclusions
In summary, optimized culture conditions were developed
for the fermentation of the recombinant E. coli which
harbored the nitrilase MG. Using the whole-cell E. coli/
MG as catalyst, a bioprocess was developed to completely
hydrolyze as high as 164 g/L phenylacetonitrile, leading to
production of 163.4 g/L phenylacetic acid. The bioprocess
could be easily scaled up and showed high potential for
industrial application.
Mihal M, Cervenansky I, Markos J, Rebros M (2017) Bioproduction
of phenylacetic acid in airlift reactor by immobilized Glu-
conobacter oxydans. Chem Pap 71:103–118. doi:10.1007/
s11696-016-0062-y
Vergne-Vaxelaire C, Bordier F, Fossey A, Besnard-Gonnet M,
Debard A, Mariage A, Pellouin V, Perret A, Petit JL, Stam M,
Salanoubat M, Weissenbach J, De Berardinis V, Zaparucha A
(2013) Nitrilase activity screening on structurally diverse
substrates: providing biocatalytic tools for organic synthesis.
Adv Synth Catal 355:1763–1779. doi:10.1002/adsc.201201098
Wang MX (2015) Enantioselective biotransformation of nitriles in
organic synthesis. Acc Chem Res 48:602–611. doi:10.1021/
ar500406s
Acknowledgements This work was supported by the National Nat-
ural Science Foundation of China (Grant No. 21406068/B060804),
the Fundamental Research Funds for the Central Universities.
Wang HL, Sun HH, Wei DZ (2013) Discovery and characterization of
a highly efficient enantioselective mandelonitrile hydrolase from
Burkholderia cenocepacia J2315 by phylogeny-based enzymatic
substrate specificity prediction. BMC Biotechnol 13:14. doi:10.
1186/1472-6750-13-14
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